Fortification of cocoa semi-skimmed milk formulations with native lactic acid bacteria: Cell viability, physicochemical and functional properties for developing novel foods

Abstract

This study aimed to evaluate several cocoa semi-skimmed milk formulations (CSMFs) as potential carriers of native lactic acid bacteria (LAB) strains to obtain novel probiotic beverages (PBs) with improved technological and functional characteristics, and satisfactorily organoleptic acceptance. The viability of two native LAB (Lactiplantibacillus plantarum UTNGt2 and Lactiplantibacillus pentosus UTNGt5) was assessed in comparison with two references (Lactococcus lactis subsp. lactis ATCC11474 and Limosilactobacillus reuteri DSM17938) strains in supplemented CSMFs throughout storage with refrigeration. The optimum conditions to produce novel beverages supplemented with native LAB were pH 6.6, 42°C, and 1 h of fermentation. Moreover, the effect of LAB strains fortification on pH, titratable acidity, total solids (°Brix), total polyphenolic compounds (TPC), antioxidant capacity (AOX), and ascorbic acid content (AAC), total proteins and fat, at initial and final storage was evaluated. The addition of two native LAB strains did alter the physicochemical quality of CSMFs to a lesser extent, where the bioactive molecules improved significantly (p < 0.05) with the increase of cocoa concentration and depending on the supplied strain. Although a statistically significant (p < 0.05) decrease in cell counts was recorded during storage, the LAB cells were found to be viable up to 21 days of storage at 4°C (>6 logCFU/ml), which is sufficient in number to prove their stability in vitro. Overall organoleptic results suggested that LAB supplementation had a significant impact on sensory attributes with satisfactory acceptability (>78%) of PBs containing the native strains and 1-2% cocoa, while CSMFs counterparts were less appreciated (40%) as perceived off-flavor. It appears that supplying bacteria to CSMF preserves flavor in the final product. Furthermore, the final beverages were free of harmful bacteria; thus, they comply with consumer safety regulations. This study concludes that CSMF can be used as a carrier of native LAB strains, maintaining cell viability, unaltered physicochemical properties, and improved functional and sensory characteristics, for which final beverages can be regarded as functional food. From the application standpoint, these formulations are an alternative to delivering native LAB strains and could help the cocoa and dairy industry to develop more attractive products for the growing regional market.

Keywords: antioxidant activity; cocoa; lactic acid bacteria; polyphenols; probiotics.

FIGURE 1

Workflow of the manufacturing of cocoa-based semi-skimmed milk beverage supplied with LAB strains. Legend: LAB, lactic acid bacteria; CSMF, semi-skimmed milk + 1–3% cocoa + 2% glucose; TPC, total polyphenol content (equivalent milligrams of gallic acid (GAE)/L); AOX, antioxidant capacity (equivalent μmol Trolox/L); AAC, ascorbic acid (equivalent mg acid ascorbic/L).

FIGURE 2

Cell viability (logCFU/ml) comparison of PBs beverages obtained without fermentation (0 h) and fermentation step (1 h). Bars are the means ± standard error. Values with different letters are significantly different p < 0.05. Capital letters show the differences within the method (Duncan’s test). Small letters show the differences within the formulation-with and without cocoa (LSD with Bonferroni correction). Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose.

FIGURE 3

Log reduction (%) in the cell counts in PBs at the end of storage (day 21) obtained with fermentation step. Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose.

FIGURE 4

Total solids (°Brix) comparison between the PBs at day 21 of storage. Bars are the means ± standard error. Values with different letters are significantly different p < 0.05. Small letters show the difference between the beverages (LSD with Bonferroni correction); Capital letters indicate the differences within strain (Duncan’s test). Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose.

FIGURE 5

Biplot PCA analysis of five variables (pH, titratable acidity, °Brix, total protein content and fat) of PBs and CSMFs at day 21 of storage; the rectangles marked the close-related beverages. Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose.

FIGURE 6

Effect of bacteria on TPC (A), AOX (B), and AAC (C) at the end of storage (21 day). Bars are the means ± standard error. Values with different letters are significantly different p < 0.05. Small letters show the difference between the beverages (LSD with Bonferroni correction); Capital letter show the differences within the beverage group (Duncan’s test). Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose. AOX, antioxidant capacity (equivalent μmol Trolox/L); TPC, total polyphenol content [equivalent milligrams of gallic acid (GAE)/L]; AAC, ascorbic acid (equivalent mg acid ascorbic/L).

FIGURE 7

Biplot PCA analysis of the nine variables (pH, titratable acidity, Brix, TPC, AOX, AAC, protein, and fat) of PBs obtained with the four probiotic strains. The green and red rectangles represent the PBs at day 1 and 21, respectively. Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose; C1–C2: values registered at 1 and 21 days for each variable. TPC, total polyphenol content; AOX, antioxidant activity; AAC, acid ascorbic content.

FIGURE 8

Radar plot of the overall acceptance of the PBs compared with CSMFs and SMs at day 21. Legend: PB1–PB4: (CSMF1–CSMF4) + UTNGt2; PB5–PB8: (CSMF1–CSMF4) + UTNGt5; PB9–PB12: (CSMF1–CSMF4) + L.Lac; PB13–PB16: (CSMF1–CSMF4) + L.r. CSMF1: semi-skimmed milk + 1% cocoa + 2% glucose; CSMF2: semi-skimmed milk + 2% cocoa + 2% glucose; CSMF3: semi-skimmed milk + 3% cocoa + 2% glucose; CSMF4: semi-skimmed milk + 2% glucose; SM1: semi-skimmed milk + 1% sterile distilled water + 2% glucose; SM2: semi-skimmed milk + 2% sterile distilled water + 2% glucose; SM3: semi-skimmed milk + 3% sterile distilled water + 2% glucose.