NON-DAIRY FERMENTED MILK PRODUCTS a very detailed study

By Tasneem Gufrana

1. NON-DAIRY FERMENTED MILK PRODUCTS U n de r t h e Gu i da n ce of Dr. Roji B. Waghmare Department of Food Engineering and Technology I ns t i t u t e of Ch e m i cal T e chn o l o g y Mumb ai Pr e s e n t e r : TASNEEM GUFRANA R o l l N o . : 23FBT210

2. CONTENTS

3. INTRODUCTION Non-dairy fermentation refers to the biological transformation of plant-based substances with microbes, such as cereals, legumes, and nuts, into cultured and fermented products that mimic the texture, flavor , and nutritional properties of traditional dairy products ( Tangyu et al., 2019) Leads to : - the development of unique flavors - enhanced nutritional profiles, and - improved digestibility Need for plant-based milk alternatives: - free from lactose, so they are good for people who are lactose intolerant (Crittenden and Bennett 2005), - also free from dairy allergens such as casein - low in saturated fat, hence provides a better option for people looking for lower-fat alternatives - consumer need for vegan-friendly labels (Hughes 1995, Craig 2010) - has better carbon footprint as the production is related to less emission of GHGs per gram of protein ( Rotz et al. 2010) Table 1. Carbon footprint for vegan Vs normal milk (adapted from Sunidhi et al., 2021) Milk type Greenhouse Gas Emissions (kg - Cow milk 0.62 Rice milk 0.23 Soy milk 0.21 Oat milk 0.19 Almond milk 0.16 Milk type Cow milk 0.62 Rice milk 0.23 Soy milk 0.21 Oat milk 0.19 Almond milk 0.16

4. INTRODUCTION The rapid growth and global acceptance of plant-based dairy industry - Over the years, the global market for these products has become a multi-billion dollar business and will reach a value of approximately 26 billion USD within the next 5 years. - The Western countries have started accepting plant based milk to a great extent. Hence, the plant based milk makes up to 15% of the total milk industry. - The plant-based dairy industry is projected to grow at a Compound Annual Growth Rate (CAGR) of 12.5%, reaching a worldwide market valuation of approximately USD 52.58 billion by the year 2028 ( Tangyu et al., 2019) Alternate names for plant-based dairy products Plant-based alternatives Vegan substitutes Plant based aqueous extracts (PBAEs) Figure 1. Alternative names of vegan dairy products

5. PROPERTIES OF PLANT-BASED MILKS The commercially available milk alternatives are derived from a variety of plant sources, including legumes, seeds, nuts, cereals, and pseudo-cereals ( Mäkinen et al., 2016). Plant-based dairy substitutes can be categorized into two main groups: ( Jeske et al., 2018, Kamath et al., 2022) Dairy alternatives, in which plant-based ingredients serve as substitute carriers for nutrients and probiotics; Dairy analogues, where plant-based materials undergo transformation to replicate the taste, texture, appearance, and frequently, the nutritional profile of authentic dairy products. Advantages of plant-based milk alternatives Legumes and seeds possess a protein content distinct from that of cow milk, though the amino acid quality differs. Abundant in micronutrients (vitamins and minerals) and contain bioactive compounds like antioxidants and dietary fibers ( Gernand et al., 2016, Zhao et al., 2014) Phytoestrogens are linked to a reduced risk of osteoporosis, heart disease, breast cancer, and menopausal symptoms ( Patisaul et al., 2010) β-glucans play a role in promoting health benefits, such as lowering cholesterol levels, and enhance the sensory characteristics of the end products (Othman et al., 2011)

6. A COMPARATIVE OVERVIEW OF DIFFERENT PLANT BASED MILK ALTERNATIVES Table 2. Different composition of macronutrients, functional properties and few limiting characteristics of plant based sources as milk alternatives (adapted from Tangyu et al., 2019)

7. QUALITY PARAMETERS OF PLANT BASED MILK ALTERNATIVES Figure 2. Different parameters to be considered before choosing a plant based material (adapted from Tangyu et al., 2019) Many commercially available plant-based milk alternatives lack nutritional balance and do not match the nutritional profile of animal milk. only selected soy-based milk analogues reach the higher protein level of cow milk (3.7%) ( Jeske et al., 2017) Plant proteins frequently demonstrate low quality, limited digestibility, and an undesirable deficiency in essential amino acids Plant proteins can be challenging to digest due to the presence of antinutritional factors like protease inhibitors and non-starch polysaccharides Plant phenols, and flavonoids, contribute to bitter, pungent, or astringent flavors , influenced by their molecular weights ( Drewnowski et al., 2000)

8. FERMENTATION OF PLANT-BASED MILKS The fermentation of plant based milk is mainly done by Lactic acid bacteria (LAB), bacilli and yeasts ( Saccharomyces ) ( Jeske et al., 2018) Investigated predominantly as mono-cultures , these microbes have demonstrated capabilities that enhance crucial nutritional and sensory characteristics of plant-based products. The beany flavour of some legumes can be decreased by fermentation because of elimination of n-hexanal and n-hexanol. Thus, sensory profile is maintained (Wang et al., 2003) During cereal based fermentation, elimination of diacetyl (2,3-butanedione) is done to retain desirable flavour (Peyer et al., 2016) The benefit of LAB ( Tangyu et al., 2019): - makes the medium acidic, thus prevents the growth of some microbe - release organic acids, bacteriocins, hydrogen peroxide that can ultimately help in bio-preservation of food Food product Microbe Impact References Soy based drink Bifidobacterium (37℃, 48h) crude protein content Hou et al. 2000 Soybean meal Lactobacillus plantarum (37℃, 48h) essential amino acid (L-lysine) Song et al. 2008 Sesame milk Lactobacillus plantarum (37℃, 18h) Transform sesaminol triglucoside to bioactive sesaminolaglycone with better antioxidant properties Ulyatu et al. 2015 Table 3. Mono-culture fermentation of soy based products

9. FERMENTATION OF PLANT-BASED MILKS Favorable interactions in mixed-culture fermentation primarily involve mutualistic and commensalistic relationships, promoting beneficial activities in at least one microbe (National Research Council, 1992). Mixed cultures play a role in diminishing anti-nutrients, thereby improving mineral availability. Additionally, mixed-culture fermentation seems more effective in producing desired flavor enhancers. Food product Microbe Impact References Yoghurt Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus (37℃, 24h) Lactobacillus benefits S. thermophilus through release of aa as free N2 source and the latter benefits Lactobacillus by providing growth stimulating factors Sieuwerts et al., 2008 Peanut Lactobacillus acidophilus and L.plantarum ( 35℃, 24h) the total protein and L-lysine, L-methionine and L-tryptophan Sanni et al., 1999 Peanut milk L. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus (37℃, 24h) beany flavour whiteness, viscosity, gumminess and smoothness Lee 2001 Soy S. Thermophilus and Bifidobacterium infantis (37℃, 24h) phytic acid and saponin levels Lai et al., 2013 Table 4. Mixed –culture fermentation of different dairy and non-dairy products

10. PROCESSING OF PLANT-BASED ANALOGUE PRODUCTION The extraction of the plant-based materials to produce suitable ingredients is crucial for creating a satisfactory plant-based dairy analogue. The composition of the raw material, influenced by extraction processes, plays a significant role in determining its behavior in subsequent stages of product development. The associated pre- and post-extraction processes can result in diverse microextractions of ingredients and protein conformations. Moroever , some novel techniques can be implemented for better retention of nutrients in the final product (Jiang et al., 2013) Figure 3. Manufacturing of different plant-based milk alternatives

11. PROCESSING OF PLANT-BASED ANALOGUE PRODUCTION 1. MECHANICAL PRE-TREATMENT AND EXTRACTION Technique Conditions Plant source Process application Change in textural properties Influence in flavor Impact on nutrients References Roasting Boil for 50 min, Roast at 50 °C for 16 h Pulses ( faba bean) Roasting prior to flour production Increase in water holding capacity - Increase in total dietary fibres Ferawati et al., 2021 Dehulling Soaking in NaOH (0.07 mol/L, 10h) Peas Dehulling before aqueous extraction (yoghurt fermentation) - Reduced formation of off odorant Lower amount of extracted albumin Ma et al., 2019 Soaking and blanching Hydrated beans mixed with NaHCO3 in a thermostat water bath for 5 min Soy Blanching before aqueous extraction (yoghurt fermentation) Decreased soy protein solubility. Less firm yoghurt formed due to blanching at high temperature Reduces “beany” off flavor and chalky taste - Peng et al., 2015 Milling With free oxygen water (DO <0.3mg/L) Soy Anaerobic wet milling for aqueous extraction - Reduction in lipid oxidation products and off odour - Kaharso et al., 2021

12. PROCESSING OF PLANT-BASED ANALOGUE PRODUCTION 2. CHEMICAL PRE-TREATMENT AND EXTRACTION Technique Conditions Plant source Process application Change in textural properties Influence in flavor Impact on nutrients References pH alterations pH 4 and 0.06 mol/L NaCl Quinoa Alteration of cooking pH Regulated pH and salinity helped in 3x soluble protein extraction as compared to pure water - - Pineli et al., 2015 3. BIOLOGICAL PRE-TREATMENT AND EXTRACTION Technique Conditions Plant source Process application Change in textural properties Influence in flavor Impact on nutrients References Enzymatic α- amylase, 70 °C for 1 h. Faba bean Starch hydrolysis ( Termamyl Ultra 300 L) of slurry before yoghurt fermentation Yoghurt with higher viscosity and gel strength Yoghurt produced with higher viscosity and gel strength - Jiang et al., 2020 Germination Soaking 24 °C for 24 h and germination for 48 h Rice Germination prior to yoghurt formation (flour based) Starch hydrolysis lead to less yoghurt consistency while germination Improved sensory acceptance after fermentation. Bitterness due to lipid oxidation Increased antioxidant level and γ -aminobutyric acid Cacares et al., 2019

13. PROCESSING OF PLANT-BASED ANALOGUE PRODUCTION 4. OTHER NOVEL TECHNIQUES FOR PRE-TREATMENT AND EXTRACTION Technique Conditions Plant source Process application Change in textural properties Influence in flavor Impact on nutrients References Ultransonication 9 min high intensity ultrasound (HIU)  Soy Ultrasonication of PBAE Reduced particle size, better thermal and emulsion stability Reduction in ‘beany’ flavor and reduction in off flavor - Mu et al., 2022 High hydrostatic pressure (HHP) 281 MPa of pressure, 18.92 min of holding time, and 1:8.33 of protein isolate/water ratio. Soy Optimised HHP prior to yoghurt production Decrease in yoghurt syneresis, better protein solubility and water holding capacity Reduction in lipid oxidation activity - Wang et al., 2021 Pulsed electric field (PEF) PEF treatment at 28 kV/cm Almond Comparison with thermal processes Decreased sedimentation and increase in colloidal stability Reduction in lipid oxidation and peroxidase activity Increase in free amino acid content Manzoor et al., 2020

14. RAW MATERIALS EXPLORED FOR MILK ANALOGUE The basic raw materials that has been explored for dairy analogue come under the botanical classes of legumes, cereals/grains, and nuts/drupes/seeds The protein/fat components significantly influence the texture and taste of dairy analogs . The protein content influences: - water holding capacity - gelation - generation of flavour compounds The fat content has an effect on the: - sensory properties like mouthfeel, flavour carrying capacity Botanical classes Sub classes Protein Fat Carbohydrate Fibre Reference Legumes Soy Chickpea Faba bean Lentil Peanut 36.5 63.0 58.3 63.1 16.1 19.9 6.0 1.5 2.2 49.2 30.2 63.0 58.3 63.1 16.1 9.3 12.2 18.9 10.8 8.5 Souza et al., 2015, Cichonska et al., 2022 Grains Rice Oat Quinoa Barley 7.1 16.9 14.5 9.9 0.7 6.9 5.2 1.2 80 66.3 64.2 77.7 1.3 11.6 14.2 15.6 Petrova et al., 2020 Nuts Almond Hazelnut Cashew Walnut 21.2 15.0 18.2 15.2 49.9 60.8 43.9 65.2 51.6 16.7 30.2 13.7 12.5 9.7 3.3 6.7 Chandarsekara et al., 2016 Table 5. Different raw materials explored as milk replacements

15. FERMENTATION OF DIFFERENT PLANT MATERIALS TO IMPROVE ORGANOLEPTIC BEHAVIOUR Raw material Benchmark/Application Conditions Fermentation type Microorganisms Outcomes References Chickpea Cow’s milk 37℃, 48h Mono culture Bacillus amyloliquefacien , Lactobacillus paracasei Improved L-lysine content, removal of raffinose lead to improved digestibility, reduction in off flavor Tangyu et al., 2021 Tiger nut Milk yoghurt Pasteurization (70℃ for 30 min), starter culture (5g/L), fermentation (37℃, 8hr) Mixed culture Lactobacillus bulgaricus, Streptococcus thermophilus Increase in sprouting time lead to: Increased MC (90%) Increased protein (0.29-0.70%) Reduced fat (1.8-1.30%) Increase energy value (1.5-2%) pH reduction- suitable for probiotics Ogundipe et al., 2021 Pea, mung bean Milk yoghurt 37℃, 30 min Mixed culture Lactobacillus bulgaricus, Streptococcus thermophilus, , Lactobacillus plantarum Mung bean protein yoghurt showed better hardness, chewiness and WHC due to ds bonds and hydrophobic interactions to maintain protein gels Yang et al., 2021 Jerusalem artichoke, almond Milk yoghurt Pasteurization (85℃ for 15 sec), fermentation (40 ℃, 24h) Mixed culture Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis Increase in TPC (49.68-61.78%) Increase in WHC (81.45-89.77%) High JAM inhibited syneresis JAM (67.5%) + AM (75%) showed better properties Aydar et al., 2021

16. FERMENTATION OF DIFFERENT PLANT MATERIALS TO IMPROVE ORGANOLEPTIC BEHAVIOUR Raw material Benchmark/Application Conditions Fermentation type Microorganisms Outcomes References Coconut Milk yoghurt Pasteurization (90℃, 3 min), fermentation (43℃) Mixed culture Lactobacillus plantarum, Lactobacillus brevis Incorporation of tapioca starch (1%) led to: Reduction in syneresis Better texture 0 MC (71%), protein (2%), carb (6%), fat (20%) Pachekrepapol et al., 2020 Rice, chickpea, lentil Milk yoghurt Pasteurization (80℃, 15 min), fermentation (30℃, 16h) Mixed culture Lactoplantibacillus plantarum, Levilactobacillus brevis Energy value (67.7 kcal/100 g) Protein (3%- covered 20% of energy) Proteolysis lead to high free amino acids and release of volatile compounds Low content of phytic acid, saponins, tannins (90%) Increase in GABA content Pontonio et al., 2020 Oat Milk yoghurt Pasteurization (90℃, 30 min), fermentation (43℃, 6h Mixed culture Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus lactis, and Streptococcus thermophilus Addition of aquafaba, vegetable oil decreased syneresis, increased WHC, enhanced texture, Confocal microscopy showed gel like structure Probiotic content: S.thermophilus (>8.28 log CFU/g) and L.bulgaricus (>5.79 log CFU/g) Raikos et al., 2020

17. FERMENTATION OF DIFFERENT PLANT MATERIALS TO IMPROVE ORGANOLEPTIC BEHAVIOUR Raw material Conditions Benchmark/Application Fermentation type Microorganisms Outcomes References Pea protein isolate, olive oil fermentation (43℃) Hard cheese Mono culture Streptococcus thermophilus Lactobacillus bulgaricus Lactobacillus acidophilus, Lactobacillus paracasei , and Bifidobacterium. Higher oil lead to: weaker gel formation and increased WHC Optimal conditions: protein content (10%) and olive oil (10%) without compromising gel hardness Masia et al., 2022 Soy Sterilization (108℃, 15 min), fermentation (37℃) Soft cheese Mixed culture Lactobacillus bulgaricus, Streptococcus thermophilus, Geotrichum   candidum Increase in strain of G.candidum from 10 7 –10 8  CFU/g after 21 days at 10°C or 15°C. Degradation of soy protein and fat led to pH increase and FFA. Hardness and chewiness decreased Ripening for 28 days at 10°C showed better sensory Li et al., 2020 Oat, maize, barley Fermentation (25℃, 24h Milk kefir Mixed culture Lactobacillus plantarum, Leuconostoc mesenteroides L.plantarum + milk kefir starter in oat lead to 11.4% of RDA for 100 g Proton transfer reaction-TOF-MS showed volatile organic compound profile Yepez et al., 2019

18. CEREALS AS A MILK ANALOGUE Grains, encompassing cereals and pseudocereals, often constitute a significant portion of the basic diet in many regions due to their affordability and rich nutritional profile ( Adebo et al., 2022)

19. Oat milk is not milk but a water extract of oats, which has a smooth, milk like taste and is not only a simple plant-based nutritional drink but also contributes to a healthy lifestyle ( Bocchi et al., 2021) Commercial brands: Oatly (USA), Vitasoy (China), Alpro (UK), Pure harvest (Australia) (Xiong et al., 2022) Benefits - Has good amount of Fat, protein, vitamins, dietary fibres and a lot of micronutrients ( Jeske et al.,2018). - Helps in reduction of blood sugar, lowers cholesterol level, prevention of type II diabetes - Has better sensory than other plant based milk (almond, rice, lentil) Processing: 1. D irectly destroying and decomposing plant tissue into small particles through mechanical crushing, soaking, hydrolysis, separation, heat treatment, and homogenization unit operation, which is a relatively traditional method 2. Mixing and separating plant components, such as emulsifiers, thickeners and oils, with water, which is then heat treated and homogenized to produce an emulsion with small droplets ( Mcclements et al., 2019). Figure 4. Processing of oat based milk (adapted from Yu et al., 2023) PROCESSING OF OAT MILK

20. CASE STUDY FOR OAT MILK Objective: To explore the process optimisation to achieve maximum nutritional yield and sensory properties of oat milk by treatments such as acid, alkali, α -amylase and sprouting alone or in combination ( Babolanimogadam et al., 2023) 1. Oat milk yield and Protein extraction yield analysis: Fig: The oat milk (OM) yield and protein extraction yield of different treatments of oat milk (control (C), acid (PA), alkaline (Al), enzyme (En), sprouting ( Sp ), sprouting–acidic ( Sp -PA), sproutingenzyme ( Sp -En), enzyme–alkaline (En-Al), and acidic–enzyme (PA-En)) FINDINGS: OM yield was better for Sp , En, Sp -PA and Sp -En Yield of OM strongly affected by decreased viscosity by starch digestion and prevention of gelatiization during thermal processing and treatments with sprouting and α -amylase The PEY increased to 82.7% for Sp -PA treatment Low PEY in PA treatment is due to aggregation and precipitation of proteins in acidic conditions But in Sp -PA treatment, PEY yield was high because free amino nitrogen during sprouting may affect protein solubility

21. CASE STUDY FOR OAT MILK 2. Total phenolic content and antioxidant activity analysis: FINDINGS: TPC for PA treatment is 66 mg GAE/L for PA treatment and for En-Al is 342.67 mg GAE/L Sprouting and enzymatic treatment enhanced phenolic yield Phenolic compounds degraded under acidic conditions AOA of oat enhanced during steeping and germination. Enhancement of AOA is also due to de-novo synthesis of avenanthramides Roasting affect the extraction yield of TPC with higher AOA value Fig: The total phenolic content (TPC, mg GAE/L) and antioxidant activity (AOA, mg BHT eq /L) of different treatments of oat milk

22. NUTS AS A MILK ANALOGUE Exploring tree nuts for vegan dairy products is a fascinating area of research and development, driven by the increasing demand for plant-based alternatives to traditional dairy products Tree nuts like almonds, cashews, walnuts, and macadamias offer a rich source of nutrients, flavors , and textures that can mimic or even surpass those of animal-based dairy products. ( Shori et al., 2021)

23. PROCESSING OF ALMOND MILK Almonds are widely consumed as nut and it contains around 25% protein present as amandin . Almond has high content of vitamin E in the form of alpha-tocopherol and manganese that needs to be supplied by diets Almond seeds has been explored as a novel source of prebiotics with increased populations of bifidobacteria and Eubacterium rectale and subsequent increase in butyrate concentrations. Raw almonds Soaked for 24 hours (almond: water = 1:9) Excess water is drained and skin is removed Blend for 3 minutes Filter using 4 layer cheesecloth Almond milk Processing HPH 100-600 MPa Freeze dry 48 hours Almond milk powder stored at -20 ℃ Analysis of protein properties Figure 5. Processing of almond based milk (adapted from Bernat et al., 2015)

24. CASE STUDY FOR ALMOND MILK Objective: To develop a non-dairy, fermented probiotic product by using Lactobacillus reuteri by the combined effect of heat treatment and high homogenisation pressure on physical properties and stability of almond milk Fig: Microstructure (CLSM micrographs) and macrostructure (Canon images) of almond “milk” untreated (A), low heat treated (LH) (B), homogenised at 172 MPa (MF) (C) and treated by combined MF-LH (D) FINDINGS: The oil droplets and protein bodies dispersed in the serum phase are clearly distinguished in the microstructure of untreated milk For low heat treatment (85°C for 30 min) the majority of the almond proteins were aggregated and included oil droplets which induce formation of weak gel in macrostructure The MF3 treatment (72 MPa) reduced the size of fat globules and small particles were flocculated through protein bridge which shows poor emulsion The MF3LH (72 MPa for 121°C treatment caused the formation of big oil droplet-protein aggregates which appear embedded in a continuous protein matrix, thus enhancing the physical stability of the milk MF reduces droplet size and promotes partial protein solubilisation and heat treatment enhances protein denaturation giving rise to 3D network

25. CONCLUSION Use of proper raw materials as per nutritional value Choosing of appropriate processing parameters Fermentation increases overall nutritional value by release of bacteriocins, and different organic acids Use of biopreservation concept: bacteriocins, endolysins

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29. CRITICAL REVIEW OF RESEARCH PAPER

30. CONTENTS

31. JOURNAL METRICES PUBLICATION DETAILS AND JOURNAL DETAILS Received on 16 May 2023 Received in revised form 11 September 2023 Accepted 15 September 2023 Available online 16 September 2023 Doi (Digital object identifier) https://doi.org/10.1016/j.foodchem.2023.137511 Impact factor of journal (2023) 8.8 Cite score 14.9

32. INTRODUCTION The focus of this research is on enhancing the nutritional and functional aspects of plant-based yogurt, particularly soy yogurt. While dairy yogurt is recognized globally as a healthy food, concerns related to environmental impact, animal welfare, and allergens have propelled the popularity of plant-based alternatives. Soybean has historically been a key ingredient in yogurt production, but challenges such as allergens and limited nutritional value exist. To address these issues, the study explores the use of B. subtilis bacteria, which can enhance nutrition and reduce allergens when combined with LAB (Lactic Acid Bacteria). The incorporation of lycopene, a potent antioxidant, is also investigated for its potential health benefits. The objective of this study is to develop a multifunctional soy-based yogurt enriched with lycopene 1. Utilize B. subtilis strains genetically modified to express carotenoid genes ( crtE , crtB , and crtI ) to produce lycopene, enhancing the antioxidant properties of soy yogurt. 2. Employ a two-stage fermentation process involving B. subtilis and LAB to improve the nutritional qualities of the soy-based yogurt. 3.Evaluate the physiochemical properties, antioxidant activities, and sensory attributes of the developed lycopene-enriched soy yogurt to assess its overall functionality and acceptability.

33. MATERIALS AND METHODS 1. Plasmid maintenance and selection was done by - E.coli DH5 α in LB medium with ampicillin and chloramphenicol - B.subtilis WB800N in LB medium with chloramphenicol and glucose 2. Construction of recombinant strain - Genes crtE ( geranylgeranyl diphosphate synthase), crtB ( phytoene synthase) and crtI ( phy toene desaturase) amplified from Pantoea ananas by Phanta Max Super-Fidelity DNA Polymerase - pHT – 01-p43 was amplified as backbone - Recombinant plasmid pHT01-crtEBI mixed with competent B.subtilis cells by sudden shock treatments and positive strains selected by chloramphenicol and selected by PCR 3. Preparation of soy milk - Soybeans soaked with heavy deionized water -Soybeans filtered through a double layer of gauze to give soymilk, which is further sterilized (15 min, 105 ℃) - B.subtilis with rplasmid fermented in liquid LB medium with glucose at 37 ℃, 200 rpm for 48h - Further this combination inoculated into sterile soymilk for 12h in a shaker

34. MATERIALS AND METHODS 4. Preparation of soy yoghurt - Starter culture Streptococcus thermophilus and Lactobacillus bulgaricus added to soymilk with B.subtilis , B.subtilis with r-plasmid and without strain - Conditions: 37℃ for different time intervals 5. Determination of lycopene content: -Fermented yoghurt samples prefrozen at -20 ℃ and freeze dried at -80 ℃ for 24h -freeze dried product suspended with 1M NaOH and sonicated for 5 min -Lycopene reweighted with chloroform-methanol extract (2:1) -sample proceeded for HPLC -Lycopene standard solution made by mixing lycopene in methanol to give different concentrations for generating standard curves 6. Determination of pH and titratable acidity 7. Determination of total protein content - Analyzed by bicinchoninic acid (BCA) protein assay kit - Measured spectrophotometrically at 562 nm

35. MATERIALS AND METHODS 8. Determination of total isoflavone content - Sodium nitrite- aluminum nitrate colorimetry assay has been used for analysis -1 ml yoghurt + 0.3ml sodium nitrite + 0.3ml aluminium nitrate + 4.0ml of NaOH + 4.4ml DW -Absorbance recorded at 509 nm - Total isoflavone content calculated based on rutin concentration 9. Determination of antioxidant activity 9.1. DPPH free radical scavenging ability assay -DPPH free radical scavenging ability (% ) = where, = blank (95% ethanol) = absorbance of sample at 517 nm 9.2. Determination of ABTS free radical scavenging ability - ABTS free radical scavenging ability (% ) = where, = blank (DW) = absorbance of sample at 734 nm 9.3. Determination of ferric-reducing antioxidant power (FRAP) assay - Total antioxidant with FRAP (% ) = where, = blank (80% methanol) = absorbance of sample at 595 nm -  

36. MATERIALS AND METHODS 10. Determination of free amino acids - L-8900 automatic amino acid analyzer used - The analysis conditions for system were consisted of Hitachi ion-exchange column 2622 (4.6 mm × 60 mm, 5 μ m), injection volume of 20 μ L, and flow rate of 0.35 mL/min. - The detection wavelengths were set at 570 nm and 440 nm. 11. Sensory evaluation - Panelists received sensory evaluation training, and were asked to evaluate the color , appearance, flavor , taste, aftertaste and overall acceptability of fermented soy yogurt, as well as the comprehensive acceptance index. -

37. RESULTS AND DISCUSSIONS 1. Generation of lycopene-soy yoghurt - The lycopene peak was further confirmed in yogurt with WB800N/pHT01-crtEBI by HPLC validation - Lycopene contents were measured over time for the second phase - Lycopene components displayed a final content of 22.67 ± 2.95 mg/g DCW for fortification. - Enriched carbon sources from the soy substrate and high conductivity for enzyme expression by WB800N strain helped lycopene incorporation

38. RESULTS AND DISCUSSIONS 2. Physicochemical attributes of lycopene soy yoghurt 3. Antioxidant activity of lycopene-soy yoghurt

39. RESULTS AND DISCUSSIONS 4. Amino acid profiles of lycopene soy yoghurt 5. Sensory analysis For single fermentation: Appearance: 8.2 Aftertaste:6.5 Overall acceptability: 7 Flavor : 8.0 For dual fermentation of engineered B.subtilis with LAB : Appearance: 8.6 Aftertaste: 8.9 Overall acceptability: 9 Color : 9.5 Flavour: 8.8

40. CONCLUSION AND REFERENCES In this study, dual fermentations involving engineered B. subtilis and LAB were used to enhance a soy-based yogurt by incorporating lycopene. This approach resulted in improved pH levels, faster protein breakdown, and increased isoflavone content. The combined action of lycopene, soy, and B. subtilis led to enhanced antioxidant properties, reduced allergenic potential, and appealing taste and aroma. Essentially, this innovative method presents the lycopene-enriched soy yogurt as a promising alternative to traditional dairy yogurt, offering potential health benefits. References follow APA format as per journal guidelines

41. OTHERS The guidelines of journal have been followed here

42. CRITICAL ANALYSIS OF TITLE Given title: Enhancing the functionality of plant-based Yogurt: Integration of lycopene through dual-stage fermentation of soymilk Comments: Although the title specifies exactly about the work that has been done in the paper, still I feel it could’ve been written properly Suggested title: "Enhancing Plant-Based Yogurt Benefits via Two-Stage Fermentation and Lycopene Integration into Soymilk"

43. CRITICAL ANALYSIS OF ABSTRACT The abstract should be within 125 words. The abstract has 112 words which is within the limit Keywords are 6 in number People are increasingly turning to plant-based yogurt because it offers benefits for the environment, saves money, and is seen as a healthier choice. Soybeans are a promising option for plant-based yogurt but face challenges like bland taste, restricted nutritional value, and potential allergens when targeting health-focused consumers. “Deciphered” can be replaced with “interpreted” The full form of B.subtilis and LAB could have been given in abstract as per journal’s rules The last sentence seems vague/incomplete. It can be re-written as “The combination of engineered B. subtilis and LAB in lycopene-soy yogurt shows great promise. This blend provides tasty, hypoallergenic, and antioxidant-rich components, enhancing the benefits of plant-based yogurt”

44. CRITICAL ANALYSIS OF INTRODUCTION The first line can be re-written as, “ Within the realm of health-focused foods, more attention is directed towards optimizing balanced nutrition to facilitate medical prevention and therapeutic outcomes via functional foods” The words “admittedly demonstrated” seems unnecessary Hyphen must be provided in between words like “co-benefits, non-dairy and co-cultured”

45. CRITICAL ANALYSIS OF MATERIALS AND METHOD Full form of LB medium must be given Table S1 is not mentioned anywhere in the paper Table S2 is not mentioned anywhere in the paper Prior information about Pontoea ananas should’ve been given pHT – 01- p43 details must be given ( pHT is an expression vector with p43 promoter) Hyphen in “precooled”

46. CRITICAL ANALYSIS OF MATERIALS AND METHOD No information on the procedure for titratable acidity has been mentioned In the formula, when already “%” has been written on LHS, there’s no required to write it on RHS

47. CRITICAL ANALYSIS OF MATERIALS AND METHOD No table S3 has been mentioned anywhere in the paper There must have been labelling in the figure

48. NOVELTY AND SUGGESTIONS Novelty: Their previous study on the development of functional yoghurt rich in lycopene through Bacillus subtilis was noteworthy. And on the basis of that, they have shifted towards the rDNA concept to produce bioengineered co-culture with LAB to produce improved soy-based yoghurt is applausible . The interesting point was the improved sensory property for soy-based yoghurt with engineered co-culturing of microbe. Interesting fact: The amplification of genes in Pantoea ananas genome was done using Phanta Max Super-Fidelity DNA Polymerase. This polymerase is a superior enzyme for PCR with high fidelity having error rate 53x less than taq polymerase. It has an extension rate of 1 sec/kb. It has the ability to amplify 40 kb plasmid DNA, 20kb genomic DNA and 10kb cDNA. Suggestions: Overall paper was very interesting to me. But the introduction and abstract could’ve been written in a better way so that reader can interpret easily. Moreover, the missing components in the paper could’ve been looked into. However, I feel they must have included that in the previous published paper. Even though, they could’ve given reference

49. THANK YOU