Probiotic Flavored Fermented Goat Milk as An Adequate Vehicle for Beneficial Bacteria and Higher Total Phenolic and Antioxidant Activity

Special Article: Fermentation

Austin J Biotechnol Bioeng. 2024; 11(1): 1129.

Probiotic Flavored Fermented Goat Milk as An Adequate Vehicle for Beneficial Bacteria and Higher Total Phenolic and Antioxidant Activity

Livia Bordalo Tonucci1*, Bárbara Pereira da Silva2,4*, Maria Eliza de Castro Moreira2, Isabel Cristina Silva de Oliveira3, Mariana Rodrigues Carneiro1, Hércia Stampini Duarte Martino4, Karina Maria Olbrich dos Santos5

1UNINTA University. Rua Coronel Antônio Rodrigues Magalhães, Brazil

2Faculdade Dinamica do Vale do Piranga – FADIP Rua G, MG, Brazil

3Federal Institute of Ceará, Sobral, Brazil. Avenida Doutor Guarany, Derby Clube, Brazil

4Federal University of Viçosa. Department of Health and Nutrition, Viçosa, Minas Gerais, Brazil

5Brazilian Agricultural Research Corporation (Embrapa), Rio de Janeiro, RJ, Brazil

*Corresponding author: Livia Bordalo Tonucci Rua Coronel Antônio Rodrigues Magalhães, 359. Dom Expedito. Sobral, Ceará, Zip Code: 62.050-100. Postal code, 10, Brazil. Tel: +55 (88) 98809-9885 Email: livia_bordalo@hotmail.com

Received: January 25, 2024 Accepted: March 01, 2024 Published: March 08, 2024

Abstract

The study aimed to evaluate two grape flavored fermented goat milk produced with or without probiotics. Physicochemical characteristics, sensory analysis, antioxidant profile and probiotics viability of the dairy beverages were analyzed. The phenolic contents and antioxidant activity of probiotic milk were higher than conventional milk. A higher loss in cell viability was observed for L. acidophilus than for the B. animalis. The average sensory acceptability scores obtained by both dairy beverages were higher than 6.5. Therefore, the probiotic fermented milk showed an adequate vehicle for the probiotics with good viability, a higher total phenolic and antioxidant activity and good acceptability.

Keywords: Goat milk; Probiotic; Dairy beverage; Gastrointestinal simulation

Introduction

Probiotic functional foods are reported to provide several health benefits. Their efficacy are influenced by the selection of microbial cultures and the concentration of viable population in the product [32]. Lactobacillus and Bifidobacterium are associated with maintaining an optimum gut microbiota balance [35], decreasing the lactose intolerance symptoms [30], immune system stimulation [25], and presents antioxidative properties [26].

Probiotics have become an integral part of the complex world as biologics, pharmaceuticals, food and nutritional supplements due to their potential of providing health benefits. Currently, there is a notable increase in the consumption of non-bovine milk in substitution to conventional milk [40]. The specific nutritional composition of goat mill is related to higher protein digestibility, during digestion or technological processing, which can exert beneficial properties to the organism [37]. However, caprine milk products it is not widely accepted by consumers, mainly due to its typical flavor derived from their capric, caproic and caprylic acids content [16]. It is noted that the association of probiotic fermented milk with functional ingredients, such as juice, improves its nutritional profile as well as its sensory attributes [40]. In this way, purple grape juice stands out due to its pleasant flavor and flavonoids concentration, specifically anthocyanidins and resveratrol [13]. These compounds have important biological functions and health benefits, acting on the oxidative and inflammatory process [28].

Reactive Oxygen Species (ROS) mediated oxidative stress are known to play vital role in the development of chronic diseases such as cancer, diabetes, heart disease, stroke, Alzheimer's disease, rheumatoid arthritis, cataract and aging [3]. Thus, a novel approach is represented by the development of probiotic products exerting antioxidant activity. To neutralize the oxidant molecules, the human body synthesizes antioxidant enzymes and molecules that, together with the antioxidants contained in food, form a biological antioxidant barrier to chemicals that induce oxidative stress, either by generating ROS or by inhibiting antioxidant system [22]. Furthermore, the administration of probiotic lactic acid bacteria can modulate the gut microbiota composition and activity, influencing the metabolism of polyphenols and the release of bioactive metabolites at the intestinal level. However, there are a limited number of studies about the contribution of probiotic bacteria to the antioxidant activity of probiotic beverages [23].

The viability of probiotic microorganisms in food products and their resistance during the gastrointestinal transit is necessary to obtain health benefits related to activities exerted at the intestinal level [31]. In the production of flavored fermented milks, the addition of fruit juices or pulps may interfere in the survival of probiotic microorganisms [12], and should be investigated. Few publications on probiotic flavored fermented goat milk are available in literature [33,34,40]. Thus, the present study aimed to evaluate the viability and in vitro gastrointestinal tolerance of probiotics Bifidobacterium animalis and Lactobacillus acidophilus in flavored fermented milk produced with goat milk and grape juice, and their influence on the antioxidant activity, total phenolic content, texture and sensory features of the beverages during refrigerated storage.

Materials and Methods

Production of the Flavored Fermented Milk

The Probiotic Fermented Milk (PFM) and Conventional Fermented Milk (CFM) were produced using the commercial starter culture Streptococcus thermophilus TA-40 (Danisco, Sassenage, France; 0.003 g/100 g). The PFM was added by B. animalis subsp. lactis BB-12 and L. acidophilus La-5 (Chr. Hansen, Hørsholm, Denmark; 0.024 g/100 g). Goat milk provided by Embrapa Goats and Sheep (Sobral, Ceará, Brazil) was supplemented with 5% (w/v) sucrose and pasteurized at 90°C for 15 min, then cooled to 43±2°C for the addition of the starter and probiotic cultures. The fermentation process was conducted at 40±1°C until reaching pH 5.0±0.1. Next, the fermented milk temperature was decreased to 4°C up to the following day, and then the beverages were flavored with 20% (w/v) of purple grape juice obtained from Embrapa Grapes and Wine (Bento Gonçalves, Rio Grande do Sul, Brazil). All ingredients were mixed with a blender to form a homogeneous product. The final product was packed in polypropylene bottles and stored at 4±1°C for further analysis. The CFM was used for analysis of physicochemical properties and sensory quality in comparison with PFM. Total solids, total dietary fiber, ash, fat, and protein content were determined for PFM on the seventh day of storage (AOAC, 2012). All the analyses were performed in triplicate and were expressed as g/100g of whole matter.

Physicochemical Parameters and Instrumental Analysis

During fermentation and at 1, 7, 14, 21 e 28 days of storage at 4°C, PFM samples were taken to determine pH (pH meter Jenway 3510, Staffordshire, UK) and titratable acidity. Titratable acidity was determined according to standard methods and expressed as g/100 g lactic acid [21]. Firmness, consistency, cohesiveness and viscosity index were evaluated in CFM and PFM samples after 1, 14 and 21 days of storage using a back extrusion cell (A/BE) on a Texture Analyzer TA-XTPlus (Stable Micro Systems, Surrey, UK), as described by Buriti et al. (2014). The analyses were performed in quadruplicate.

Total Phenolic Content and Antioxidant Activity

The total phenolic compounds content in CFM and PFM were determined using the Folin-Ciocalteu method [45]. Aliquots of 0.5 mL of each fermented milk extract were added to 0.5 mL of Folin-Ciocalteu reagent (20%). After homogenization, 0.5 mL of sodium carbonate (7.5%) was added. The reaction mixture was homogenized by vortex (2865 g, 10 s) and incubated at room temperature (30 min). The reading of absorbance was performed in spectrophotometer (Thermo scientific, Evolution 606, USA) at 765 nm. Analytical curve of gallic acid (0.0 to 250 μg/mL, R2=0.999) was used to quantify the compounds. The results were expressed in mg of gallic acid equivalents/mL (mg GAE/mL). In a test tube, protected from light, aliquots of samples (PFM and CFM) were added to 1.5 mL of methanolic DPPH solution (1.1-diphenyl-2-picrylhydrazyl) and stirred by vortex (3000 rpm) for 30 s. After 30 min of standing, the absorbance of the solution was read in a spectrophotometer (Thermo scientific, 606 Evolution, USA) at 517 nm (Bloor, 2001). The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation: Scavenging ability (%) = [(control absorbance – sample absorbance) / (control absorbance)] x 100

Microbial Viability

Populations of S. thermophillus, B. animalis and L. acidophilus in PFM was determined after 1, 7, 14, 21 and 28 days of storage at 4 °C. The samples were serially diluted in sterile peptone water (1g/L) and subsequently plated, in duplicate. S. thermophilus enumeration was performed on M17 agar, containing lactose (Vetec, Duque de Caxias, Brazil; 5g/L), and incubated at 37°C for 48h. Populations of L. acidophilus were enumerated by pour plating 1 mL of adequate dilutions into MRS agar (Oxoid Basingstoke, UK), followed by incubation at 37°C for 72 h. For the selective enumeration of B. animalis, 1mL of adequate dilutions were pour plated in modified DeMan-Rogosa-Sharpe (MRS) agar (Oxoid, Basingstoke, UK), prepared with dicloxacillin (Sigma, St. Louis, US), cysteine hydrochloride (Cromoline, Diadema, Brazil), and lithium chloride (Cinética®, Jandira, Brazil) to reach a concentration of 0.5 mg/L, 0.5 g/L and 1 g/L, respectively, followed by incubation at 37°C for 72h. All bacteria were incubated in anaerobic jars (Anaerobic System Anaerogen, Oxoid, UK), except S. thermophilus, which was incubated under aerobic conditions. The results were expressed as of log colony forming units per gram (CFU/mL).

Resistance to Simulated Gastrointestinal Conditions

PFM samples were collected at 7 days of storage for the evaluation of L. acidophilus and B. animalis survival to gastric and enteric simulated conditions according to the method described by Liserre and Franco (2007), with modifications. Samples were decimally diluted in a sterile 0.85% (w/v) NaCl solution. For the gastric phase simulation, the pH was set at 2.5 with 1 N HCl solution. Pepsin (Sigma-Aldrich, St. Louis, USA) and lipase (Aldrich Chemical Company, Milwaukee, USA) solutions were added to reach final concentrations of 3.0 g/L and 0.9 g/L, respectively. The flasks were incubated at 37°C for 2 h under agitation (150 rpm). Subsequently, enteric conditions were simulated in two phases. In the enteric phase 1, the pH was increased to 5.0 with a sterile alkaline solution (150 mL of 1 N NaOH, 14 g of PO4H2Na.2H20 and distilled water up to one 1 L), and bovine bile and pancreatin (Sigma-Aldrich, St. Louis, USA) were added to reach a concentration of 10 g/L and of 1 g/L, respectively. After 2 h of incubation at the same conditions, the pH was adjusted to 6.5 - 7.0, and the respective bile and pancreatin concentrations were adjusted to 10 g/L and 1 g/L for the second enteric phase, followed by an additional incubation period of 2 h. In order to enumerate the viable L. acidophilus and B. animalis cells, aliquots were taken at the assay baseline (0 h) and after 2, 4 and 6 h and serially diluted in peptone water solution. Adequate dilutions (1 mL) were pour plated in acidified MRS agar, followed by anaerobic incubation at 37°C for 48 h. A survival ratio (SR%) was calculated based on the initial and final populations to estimate the relative resistance of each strain to the simulated TGI conditions. All results are presented as log CFU/mL.

Sensory Analysis

The sensory evaluation of the flavored fermented goat milk was approved by the Federal University of Viçosa Human Ethics Research Committee, Brazil (Process No. 219.644; CAAE: 13380413.8.0000.5153) and was carried out at the Laboratory of Sensory Analysis of UNINTA College. Sensory evaluation was performed with CFM and PFM samples after 7 days of cold storage (4±1°C) through acceptability tests, using the hybrid hedonic scale (1 = disliked extremely, 5=neither liked nor disliked, 9=liked extremely) focusing on attributes of taste, flavor, color, consistency and overall acceptability [38]. The samples were maintained under refrigeration prior the tests and served, monadically, in individual disposable plastic cups (approximately 30 mL) codified with three random digits. The sensory test was carried out with 63 untrained panelists aged 19 – 40 years old recruited among potential consumers of the beverages, and performed in individual booths. Water and unsalted crackers were available during a 1 min rest period between sample sets to refresh the palate. The consumers were also instructed to report the sensory attributes that they liked and disliked most in the samples.

Statistical Analysis

The experimental data were analyzed by SPSS software version 20 (IBM, Armonk, NY) and the results were expressed as mean±Standard Deviation (SD). Values were the average of quadruplicate/triplicate/duplicate experiments. Before analysis, data were checked for the normality, homogeneity of variances and sphericity using the Shapiro-Wilk, Levene’s and Mauchly’s tests, respectively. Differences between trials (CFM and PFM) in a single moment were tested using unpaired t test or Mann-Whitney test. Differences between experimental storage periods were statistically analyzed using repeated measures Analysis of Variance (ANOVA), followed by the post hoc Bonferroni test, taking on p<0.05. When normality was not found, the equivalent non-parametric tests were applied. Differences at p<0.05 were considered to be significant.

Results and Discussion

Composition, Physicochemical and Instrumental Analysis

Probiotic Flavored Fermented goat Milk (PFM) had the following chemical composition: total solids 17.3±0.04 g/100g, protein 2.47±0.02 g/100g, fat 2.64±0.02 g/100g, ash 0.74±0.01 g/100g and total dietary fiber 0.14±0.01 g/100g. Total solids, protein and fat contents of PFM were found to be lower than other study [29], reflecting the higher moisture content in flavored fermented milk due to the addition of grape juice. Changes in these parameters, especially total solids and fat content may affect physical-chemical properties such as viscosity, syneresis and water holding capacity [39].

The texture parameters analyse of the dairy beverages are presented in Table 1. The measured firmness, consistency, cohesiveness and viscosity index of Conventional Fermented Milk (CFM) showed an increase during the 28 days of storage, but the values registered at 14 and 28 days did not differ significantly (p>0.05). Interestingly, these parameters values increased only until day 14 (p<0.05) for PFM, however, did not affect the sensory acceptability scores. PFM presented a lower viscosity index on the first day of storage (p=0.04) compared to CFM, and no significant difference was detected for firmness and consistency (p>0.05) when the two trials were compared at the same sampling period. However, little information exists about the influence of probiotic strains on physicochemical properties of dairy beverages. Fermented milk prepared using only probiotic strains, such as L. acidophilus and Bifidobacterium spp. are often characterized by the undesirable sensory proprieties and texture [40], whereas physical properties such as firmness and ability to retain water are important factors for quality assessment [18]. As we report below, these parameters were not affected in the present study, probable due to the presence of starter culture S. thermophilus in both beverages.