Development and characterization of lactose-free probiotic goat milk beverage with bioactive rich jambo pulp

Abstract

Goat milk is considered a suitable matrix for the successful incorporation of probiotics, also obtaining new lactose-free fermented products can expand its use. This study aimed to develop and characterize formulations of lactose-free probiotic fermented goat dairy beverages as well as to determine the most appropriate concentration of red jambo pulp to be added. The beverages were developed with different concentrations of lactose-free goat milk and frozen jambo pulp (12, 15 and 18% w/v) and lyophilized (3, 6 and 9% w/v), corresponding to formulations F1 to F6, respectively, as source of bioactive compounds. Probiotics counts decreased significantly (from 8.58 to 7.38 log CFU mL⁻¹). The formulation with a higher proportion of lyophilized (F6) pulp showed the highest levels of phenolic compounds (72.08 mg GAE 100 g⁻¹), anthocyanins (50.80 mg cyanidin-3-glycoside 100 g⁻¹), ascorbic acid (41.68 mg 100 g⁻¹), and antioxidant activity (16.21 μmol TE g⁻¹) (P < 0.05). On the other hand, F3 presented the highest global acceptance and purchase intention (P < 0.05). However, the principal component analysis (PCA) indicated that the components related to bioactive compounds (PC1) stood out on sensory attributes (PC3 and PC4) and, therefore, F6 was most appropriate for obtaining a lactose-free goat probiotic fermented milk with improved bioactive properties targeting lactose intolerant consumers and those who are allergic to bovine milk proteins.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05399-z.

Introduction

The growth of the dairy sector and consumer interest in foods that provide health benefits, with adequate sensory quality, have driven the development of new products (Saqueti et al. 2019). Goat milk has a hypoallergenic effect, greater digestibility of fats and proteins and higher levels of vitamins A, B and calcium when compared to bovine milk (Delgado et al. 2020). In addition, it has the potential for the successful delivery of probiotics, expanding their use (Ranadheera et al. 2019). However, goat milk is not recommended for lactose intolerant people, lactose-free product consumption being the best alternative for them. The beta-galactosidase enzyme from Kluyveromyces lactis has been used to produce lactose-free dairy products, converting lactose into two monosaccharides (glucose and galactose), easily absorbed by the body (Dekker et al. 2019).

Probiotic cultures are used as ingredients in large-scale production of functional dairy products because of the many health benefits associated with their consumption (Al-Hindi; Ghani, 2020). In this sense, products fermented with Lacticaseibacillus paracasei (previously called Lactobacillus paracasei) presented several beneficial effects on a variety of pathologies, including gastric mucosal lesion prevention; allergy relief; reduced fat accumulation; hypocholesterolemia, antihypertensive and anti-osteoporosis effects (Lin et al. 2017); antimutagenic, antioxidant and anticancer properties (Pimentel et al. 2015); anti-inflammatory properties (Choi et al. 2020); reduction of infectious diseases (Poon et al. 2020), respiratory diseases (Nocerino et al. 2017) and liver diseases (Yao et al. 2019). Among probiotic foods, milk-based beverages are the main vehicles of these microorganisms consumed worldwide (Andrade et al. 2019).

Currently, there are several types of dairy drinks on the market that differ in terms of flavor, aroma, consistency, non-dairy ingredients used, manufacturing process, fermentation and the amount of whey used (Santos et al. 2021). The employment of whey in dairy beverage formulations is the main alternative for an adequate use of this by-product (Figueiredo et al. 2019; Souza et al. 2020), whose proteins have several functional and biological properties, such as antioxidant, antihypertensive, anticancer, antiviral, antibacterial, anti-inflammatory, and immunomodulatory activities, in addition to protecting the cardiovascular system (Costa et al. 2021). Several studies have addressed the use of whey in obtaining dairy beverages with the addition of tropical fruit such as guava and graviola (Buriti et al. 2014), papaya and orange (Andrade et al. 2019), cajá-manga (Cheuczuk et al. 2018). However, until the present moment, there are no reports in the literature on the addition of red jambo pulp to dairy beverages.

Red jambo (Syzygium malaccensis), also known as the Malay apple, considered native from Southeast Asia and Oceania, belongs to the Myrtaceae family. It has attractive color and can be used as a natural dye in foods and beverages. It’s an obovoid fruit of red with smooth skin and a white and juicy pulp, it has a similar flavor to green grapes, it is a tropical fruit characterized by significant contents of anthocyanins (12.90 mg cyanidin-3-glycoside 100 g⁻¹⁾, flavonoids (12.86 mg 100 g⁻¹), total phenolic compounds (14.81 mg GAE 100 g⁻¹) (Batista et al. 2016), ascorbic acid (171, 14 mg 100 g⁻¹) and antioxidant activity (25,92 μmol TE g⁻¹) (Nunes et al. 2016). Araújo et al. (2021) found that freeze-dried and frozen pulp of this fruit, obtained without synthetic additives, presented adequate technological characteristics for industrialization. In this regard, the addition of red jambo pulp in the formulation of probiotic fermented milk drinks can be an alternative source of such bioactive compounds and important for the incorporation of the fruit in large scale industrial applications. Moreover, until the present moment, this research was the first to report the successful application of Lacticaseibacillus paracasei associated with starter culture in a lactose-free goat dairy beverage with the addition of red jambo pulp.

Given this context, the objective of this investigation was to develop and characterize, through physicochemical, microbiological, and sensory analyses, different formulations of lactose-free goat probiotic fermented milk during refrigerated storage as well as to determine the suitable concentration of jambo pulp to obtain a bioactive enriched beverage. This will serve to expand the offerings of probiotic bioactive products based on milk and fruit, aimed at two groups of consumers with special needs: those who are lactose intolerant and those who are allergic to bovine milk proteins.

Materials and methods

Lactic and probiotic cultures

Lacticaseibacillus paracasei (formerly named Lactobacillus paracasei) (BGP 1), Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Y450B, 5 UC) were obtained from SACCO Brasil (Campinas, São Paulo, Brazil). The inoculum was prepared by diluting 1 g of the cultures in 250 mL of goat milk (previously pasteurized at 90 °C, treated with 0.07% v/v beta-galactosidase (Prozyn, São Paulo, Brazil) from Kluyveromyces lactis yeast with lactase activity of 50,000 ONPGU/g for 5 h, and submitted to a new heat treatment at 85 °C for enzymatic inactivation) and incubation at 42 °C/2 h.

Obtaining and processing raw materials

Goat milk and red jambo were purchased at the Academic Unit, Specialized in Agricultural Sciences at the Rio Grande do Norte Federal University, Macaíba—RN Campus, Brazil. The pulps were obtained from fruit that was cleaned in running water, sanitized in chlorinated water at 100 ppm for 15 min and drained to remove residual water. Subsequently, the pulps were cut longitudinally to remove the seed, fractioned with peel (rectangular shape of approximately 3 × 4.5 cm) and pulped in a semi-industrial fruit pulper (Itametal, Itabuna, Bahia, Brazil), weighed and divided into two portions: one portion was lyophilized, and the other was packed in 150 g nylon-polyethylene packages (pouch-type packaging, 12 × 20 cm transparent with non-toxic, odorless side sealing) and stored in a horizontal freezer (Electrolux, São Paulo, Brazil) at − 18 °C.

The lyophilization was done by placing 100 g of fresh jambo pulp in 18 cm stainless steel trays and storing at  − 18 °C/48 h. The trays were transferred to the lyophilizer (Terroni, São Paulo, Brazil) at − 30 °C for 48 h, constant speed of 1 mm/h, and vacuum ranging from 80 µHG to 200 µHg. The powder obtained was packed in nylon-polyethylene packages (with previously described properties), vacuum sealed, and stored at room temperature (28 ± 1 °C), in a second metallized polyethylene package (silver stand up pouch, 12.5 × 19.5 cm) with hermetic closure, protected from light.

Frozen and lyophilized pulps presented, respectively, 91.09 and 8.75% moisture (determined in an oven at 70 °C/6 h); 0.71 and 2.02% protein; 0.45 and 1.98% lipids; 0.23 and 4.60% ash; reducing sugars (3.21 and 42.53%); soluble solids (7.70 and 80%); pH (3.36 and 3.67) and 0.941 and 0.259 water activity (Aw).

Obtaining lactose-free goat milk and whey

Fresh goat milk was pasteurized at 75 °C/15 s, cooled to 10 °C and added to 0.07% v/v beta-galactosidase. After incubation at 10 °C/5 h, hydrolyzed goat milk was heat treated at 85 °C to inactivate the enzyme. Goat whey was obtained from the production of curd cheese. The whey hydrolysis, after heat treatment at 65 °C and cooling at 10 °C, received of 0.07% v/v beta-galactosidase. After incubation at 10 °C for 5 h, the hydrolyzed goat whey was heat treated at 65 °C for 30 min to inactivate lactase, followed by cooling to 10 °C. The lactose content was evaluated as described by Burgner and Feinberg (1992) by high-performance liquid chromatography using an Agilent Technologies liquid chromatograph (Infinity 1260, USA) with an automatic sampler (injection of 20 µL), quaternary solvent system, column oven and evaporative light scattering detector (ELSD). Lactose was not detected in goat whey or milk with the addition of beta-galactosidase, which were used later in the formulation of dairy beverages.

Goat dairy beverage processing

Six formulations of probiotic dairy beverages were made using lactose-free goat whey and milk, sucrose (Alegre, Mamanguape, Brazil), stabilizing mixture (65% modified starch and 35% gelatin) (Gemacon Tech, Natal, Brazil), starter culture and probiotic culture mentioned earlier and red jambo pulp. The formulations consisted of variations in the concentrations of frozen, lyophilized jambo pulp and lactose-free goat milk. In addition, a control formulation (F0) was developed in which no pulp was added to assess the effects on the viability of the probiotic. The proportion of ingredients used in lactose-free probiotic goat dairy beverage formulations with the addition of jambo pulp (frozen and lyophilized) are shown in Table 1.

Table 1.

Proportion of ingredients used in lactose-free probiotic fermented goat dairy beverage formulations with the addition of red jambo pulp (frozen and lyophilized)

Formulations
%	F0	F1	F2	F3	F4	F5	F6
Sucrose	10.00	10.00	10.00	10.00	10.00	10.00	10.00
Stabilizing mixture	0.60	0.60	0.60	0.60	0.60	0.60	0.60
L. paracasei	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Starter culture	1.00	1.00	1.00	1.00	1.00	1.00	1.00
1LJ Pulp	–	–	–	–	3.00	6.00	9.00
2FJ Pulp	−	12.00	15.00	18.00	–	–	–
3LFGM Whey	40.00	40.00	40.00	40.00	40.00	40.00	40.00
4 LFG Milk	47.90	35.90	32.90	29.90	44.90	41.90	38.90

¹ Lyophilized jambo pulp; ² Frozen jambo pulp; ³ Lactose-free goat milk whey; ⁴ Lactose-free goat milk. F0 (control/without pulp); F1, F2 and F3 (dairy beverage with addition of 12%, 15% and 18% of frozen red jambo pulp); F4, F5 and F6 (dairy beverage with addition of 3%, 6% and 9% lyophilized red jambo pulp)

The mixture was homogenized, pasteurized (65 °C/30 min) and cooled to 42 °C, at which the previously prepared starter culture (0.5% v/v) and probiotic (1% v/v) inoculants were added. The obtained milk bases were incubated at 42 °C until reaching a pH value of 4.6 (approximately 4 h). Subsequently, they were cooled to 10 °C and different concentrations of frozen pulp, 12%, 15% and 18% w/v, that were previously thawed under refrigeration temperature at 5 °C/15 h and lyophilized pulp, 3%, 6% and 9% (w/v), corresponding to formulations F1, F2, F3, F4, F5 and F6, were added respectively. The beverages were packaged in sterile polyethylene packaging, with a capacity of 900 mL and a pink color and stored at 5 °C for 28 days for further analysis to be carried out at different time intervals (1, 7, 14, 21 and 28 days) (Fig. 1).

Fig. 1.

Header: Flowchart of the production of different lactose-free goat probiotic milk beverage formulations with addition of red jambo pulp

Physicochemical and bioactive compounds analyses of formulations

The different formulations of dairy beverages were initially characterized through the analysis of lipids (Aoac 2000.18), proteins (Aoac 939.02), reducing sugars (Aoac 923.09), ash (Aoac 930.22) and moisture (Aoac 950.46). During refrigerated storage (1, 7, 14, 21 and 28 days), analyses of the pH (Aoac 947.05), total acidity (Aoac 920.124), ascorbic acid (Aoac 967.21) (AOAC 2005) and syneresis index by the drainage method were performed. All analyses were performed in triplicate.

Evaluation of bioactive compounds and antioxidant activity of formulations

Obtaining extracts

The determination of total phenolic compounds, total monomeric anthocyanins and yellow flavonoids was carried out based on preliminary tests, diluting 5 g of formulations made with frozen and lyophilized pulp in 25 mL ethanol/distilled water (70:30 v/v) (Neon, São Paulo, Brazil). After homogenization, the extracts were placed in 50 mL centrifuge tubes where they remained protected from light at 8 ± 2 °C for 24 h. After this period, the extracts were filtered on qualitative filter paper and later analyzed.

Determination of total phenolic compounds, total monomeric anthocyanins, and yellow flavonoids

The total phenolics content was determined using the Folin-Ciocalteu method and the results were expressed in milligrams of gallic acid equivalents per hundred grammes of the sample (mg GAE 100 g⁻¹). The content of total monomeric anthocyanins was determined by the pH difference, as described by Giusti and Wrolstad (2001) and calculated by the following equation:

$$\text{AMT}=\text{A}\times \text{MW}\times 1000/\epsilon \times \text{C})$$

where A: absorbance (A₅₁₀nm−A₇₀₀nm) pH 1,0-(A₅₁₀nm−A₇₀₀nm) pH 4,5; MW: molecular weight of cyanidin-3-glycoside = 490 g mol⁻¹; ε: molar absorptivity of cyanidin-3-glycoside = 26,900 M⁻¹ cm⁻¹; C: buffer concentration (mg mL⁻¹).

The analysis of yellow flavonoids was performed according to the methodology proposed by Francis (1982) and the results were expressed as milligrams per hundred grammes of the sample (mg 100 g⁻¹). All analyses were performed in triplicate using a digital spectrophotometer (model 722 N, Edutec, São Paulo, Brazil) during the entire storage period of the beverages (1, 7, 14, 21 and 28 days). The antioxidant activity was determined through the ability of the antioxidants, present in the samples, to deactivate the stable radical DPPH •, according to the methodology described by Brand-Williams et al. (1995) and the results were expressed as µMol Trolox equivalent antioxidant capacity per 100 g (Sigma-Aldrich, São Paulo, Brazil).

Microbiological analysis of dairy beverage formulations

The formulations were initially submitted to total coliforms analysis at 45 °C (NMP mL⁻¹) and to Salmonella sp. detection (APHA 2001), while the viability of the lactic bacteria was determined during the entire beverage storage period (1, 7, 14, 21 and 28 days). Lacticaseibacillus paracasei was grown in MRS agar culture medium plus sodium propionate (0.3% w/v) and lithium chloride (0.2% w/v) in anaerobiosis at 37 °C/72 h. Lb. delbrueckii subsp. bulgaricus was grown in MRS agar medium in anaerobiosis at 45 °C/48 h, and Streptococcus thermophilus was grown on the surface of M17 agar at 45 °C/24 h under aerobic conditions.

Sensory analysis

This research was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (2.063.798/CAAE: 65937117.3.0000.5537). The different formulations were submitted to the acceptance and purchase intention tests during refrigerated storage (1, 7, 14, 21 and 28 days), with a mean of ≥ 7.0 (hedonic scale) considered as global acceptance criteria.

The purchase intention was assessed using a five-point scale (5 = certainly would buy; 1 = certainly would not buy) and the attributes, color, aroma, consistency, flavor, and global acceptance were assessed through an affective laboratory test using a hedonic scale of nine points (9 = liked very much, 1 = disliked very much). During the entire storage period, the evaluations of the drinks were performed with the same non-trained judges (n = 57), of both genders, aged from 19 to 49 years old. Both sensory tests were performed with approximately 25 mL of the formulations served in disposable 50 mL cups encoded with random three-digit numbers. The judges were offered cream crackers and mineral water to eliminate residual tastes between samples.

Experimental design and statistical analysis

The experiments were performed using a completely randomized design (CRD), and the results were subjected to analysis of variance (ANOVA) with comparison of means using Tukey’s test at a 5% significance level.

Principal component analysis (PCA) was performed with the bioactive compounds and the sensory parameters of the 1st and 28th days of storage through the methodological precepts established by Sneath and Sokal (1973) to assist in defining the most appropriate pulp concentration for obtaining beverages. The main components were obtained through the correlation matrix using the varimax orthogonal rotation method to give the factors greater potential for interpretability. The Statistica version 7.0 software (Statsoft, Tulsa, EUA) was used.

Results and discussion

Physicochemical characterization of formulations

The highest levels of lipids, proteins and reducing sugars were observed in beverage formulations with higher concentrations of goat milk, while the ash contents were higher in formulations with a higher concentration of lyophilized pulp (Table 2). These results were expected considering that fruit are usually a source of minerals, they have high water activity and low content of proteins and fat. Similar levels of lipids and ashes and lower levels of proteins were previously reported in a goat probiotic dairy beverage with the addition of guava pulp (Buriti et al. 2014).

Table 2.

Physicochemical characterization¹ of lactose-free probiotic fermented goat dairy beverage formulations with the addition of red jambo pulp (frozen and lyophilized)

Formulations
	F1	F2	F3	F4	F5	F6
Moisture	80.77b ± 0.01	81.31a ± 0.03	81.74a ± 0.03	78.65c ± 0.33	77.59d ± 0.22	75.22e ± 0.2
Reducing sugars	17.09ab ± 0.05	15.00ab ± 0.05	13.86b ± 0.06	18.55a ± 2.78	16.25ab ± 1.61	14.14ab ± 1.7
Proteins	2.40c ± 0.02	2.22d ± 0.11	2.04e ± 0.02	2.95a ± 0.04	2.83ab ± 0.06	2.74b ± 0.03
Lipids	1.26b ± 0.05	1.22b ± 0.01	1.17b ± 0.01	1.47a ± 0.08	1.44a ± 0.03	1.41a ± 0.10
Ash	0.45d ± 0.02	0.46d ± 0.01	0.47d ± 0.03	0.59c ± 0.01	0.77b ± 0.01	0.87a ± 0.02

¹ Results expressed as a percentage (mg 100 g⁻¹) F1, F2 and F3 (dairy beverage with 12%, 15% and 18% frozen red jambo pulp addition); F4, F5 and F6 (dairy beverage with 3%, 6% and 9% lyophilized red jambo pulp addition). Different lowercase letters on the same line indicate significant differences between formulations (P < 0.05). Mean ± standard deviation, n = 3

Formulations F4, F5 and F6 showed significantly higher levels of protein, fat, and ash than F1, F2 and F3 (Table 2) because of the different pulp compositions subjected to freezing or lyophilization. Likewise, Bezerra et al. (2015) reported higher values of reducing sugars and ash in frozen goat probiotic yogurt with the addition of powdered jambolan fruit pulp than with the addition of frozen jambolan fruit pulp.

The moisture content of formulations made with frozen jambo pulp was higher with increasing concentration (F3 > F2 > F1), while the opposite was observed for drinks formulated with lyophilized pulp (F6 < F5 < F4). Moreover, F1 to F3 had a higher moisture content than F4 to F6 (Table 2); it was expected and derived from the process of obtaining the lyophilized pulp (8.75% w/w moisture in relation to 91.09% w/w moisture in the frozen pulp) (Araújo et al. 2021).

The pH values decreased, and the total acidity increased during storage according to the different concentrations of goat milk and the type and percentage of pulp added (Table 3), which was also reported by Gomes et al. (2013) in goat milk beverages formulated with the addition of guava gelatin during storage and by Vénica et al. (2016) in probiotic yogurt with a reduced lactose content. It is interesting if the results do not alter the sensory quality and technological characteristics of the beverages, as higher acidity values inhibit the development of deteriorating microorganisms, prolonging the shelf life of the product (Costa et al.2017).

Table 3.

pH values, total acidity and syneresis index (% w/w) of lactose-free probiotic fermented goat dairy beverage formulations with the addition of red jambo pulp (frozen and lyophilized) during 28 days of refrigerated storage

		1 day	7 days	14 days	21 days	28 days
pH	F1	4.58Aa ± 0.06	4.54ABa ± 0.02	4.51Ba ± 0.01	4.30Ca ± 0.02	4.25Ca ± 0.07
	F2	4.56Aab ± 0.01	4.52ABa ± 0.02	4.50Ba ± 0.03	4.24Cb ± 0.02	4.17Dab ± 0.02
	F3	4.54Ab ± 0.13	4.52Ba ± 0.07	4.50Ba ± 0.06	4.19Cc ± 0.03	4.10Dbc ± 0.01
	F4	4.46Ac ± 0.01	4.32Bb ± 0.03	4.26Cb ± 0.01	4.21Dbc ± 0.01	4.11Ebc ± 0.01
	F5	4.42Ad ± 0.01	4.29Bbc ± 0.01	4.21Cc ± 0.06	4.12 Dd ± 0.02	4.08Dbc ± 0.03
	F6	4.30Ae ± 0.01	4.26 Ac ± 0.01	4.18Ad ± 0.01	4.05Be ± 0.06	4.01Bd ± 0.10
Total acidity	F1	0.55Dd ± 0.01	0.57Cd ± 0.05	0.60Bd ± 0.01	0.61Ad ± 0.01	0.62Af ± 0.00
	F2	0.56Bd ± 0.01	0.58Bd ± 0.05	0.62Ac ± 0.01	0.62Ad ± 0.05	0.64Ae ± 0.01
	F3	0.60Cc ± 0.02	0.62BCc ± 0.01	0.64Bc ± 0.01	0.67Ac ± 0.01	0.68Ad ± 0.05
	F4	0.65Db ± 0.01	0.66CDb ± 0.01	0.68BCb ± 0.01	0.70ABb ± 0.01	0.71Ac ± 0.01
	F5	0.67Db ± 0.01	0.69Ca ± 0.01	0.71BCa ± 0.01	0.72ABab ± 0.01	0.74Ab ± 0.01
	F6	0.71Ba ± 0.01	0.71Ba ± 0.01	0.72Ba ± 0.01	0.73Ba ± 0.01	0.76Aa ± 0.09
Syneresis index	F1	47.50Aa ± 1.29	45.70ABa ± 0.53	42.32CDb ± 1.16	44.25BCb ± 0.12	41.09Db ± 1.01
	F2	49.87Aa ± 1.59	47.01Ba ± 1.00	43.90Cab ± 0.66	46.63Ba ± 0.57	47.43ABa ± 0.51
	F3	50.14Aa ± 1.62	48.33ABa ± 2.08	45.23Ca ± 1.08	47.53ABa ± 0.50	48.27ABa ± 1.42
	F4	26.90Ab ± 1.82	26.17Ab ± 1.76	26.08Ac ± 0.41	25.57Ac ± 0.51	25.93Ac ± 0.85
	F5	24.57Ab ± 0.51	24.67Ab ± 1.52	23.13ABd ± 0.81	22.67ABd ± 0.58	20.78Bd ± 1.06
	F6	16.67Ac ± 1.53	16.60Ac ± 0.69	15.77Ae ± 1.36	16.40Ae ± 0.53	15.17Ae ± 1.30

F1, F2 and F3 (dairy beverage with addition of 12%, 15% and 18% of frozen red jambo pulp); F4, F5 and F6 (dairy beverage with addition of 3%, 6% and 9% lyophilized red jambo pulp). Different capital letters on the same line indicate significant differences between the storage periods of the drinks. Different lowercase letters in the same column indicate significant differences between formulations (P < 0.05). Mean ± standard deviation, n = 3

F1, F2 and F3 beverages showed a higher syneresis index (P < 0.05) than formulations F4, F5 and F6 (Table 3), possibly related to the type and amount of pulp added to the beverages and considered satisfactory when compared to the syneresis index obtained by Bezerra et al. 2012 in yogurts prepared from mixtures of goat and buffalo milks. Formulations F1, F2, F3 and F5 showed a significant reduction in syneresis over storage time (Table 3), already reported in probiotic goat yogurt (Silva et al. 2017).

Bioactive compounds and antioxidant activity

Beverages F4, F5 and F6 presented a higher concentration of total phenolic compounds, total anthocyanins, yellow flavonoids, and ascorbic acid than formulations made with frozen jambo pulp (Table 4), which demonstrates that the lyophilization process was effective, preserving the bioactive compounds present in the pulp.

Table 4.

Content of total phenolic compounds *, total anthocyanins **, yellow flavonoids ***, and ascorbic acid *** present in lactose-free probiotic fermented goat dairy beverage formulations with the addition of red jambo pulp (frozen and lyophilized) during 28 days of refrigerated storage

		1 day	7 days	14 days	21 days	28 days
Total phenolic compounds	F1	41.50Af ± 1.41	39.59Ad ± 0.99	35.46Bd ± 1.99	35.34Bf ± 0.13	33.23Be ± 0.22
	F2	44.76Ae ± 0.55	40.73Bcd ± 0.77	37.55Cd ± 1.00	34.27De ± 0.15	32.04Ee ± 0.01
	F3	47.63Ad ± 0.29	42.57Bc ± 1.00	41.63Bc ± 0.84	38.67Cd ± 1.20	37.42Cd ± 0.68
	F4	67.14Ac ± 0.03	62.97Bb ± 0.98	61.10Cb ± 0.20	59.53Dc ± 0.47	59.06Dc ± 0.03
	F5	69.38Ab ± 0.30	67.13Ba ± 0.33	67.30Ba ± 0.10	63.83Cb ± 0.32	61.17Db ± 0.17
	F6	72.08Aa ± 0.98	68.31Ba ± 0.35	68.11Ba ± 0.88	67.45Ba ± 0.75	66.03Ba ± 0.97
Total anthocyanins	F1	8.48Ae ± 0.25	8.41Ade ± 0.31	7.44Be ± 0.22	6.27Df ± 0.21	6.13Dd ± 0.09
	F2	8.97Ae ± 0.12	7.89Be ± 0.09	7.83Bde ± 0.01	7.03Ce ± 0.17	6.60Dd ± 0.20
	F3	10.04Ae ± 0.03	9.03Bd ± 0.47	9.01Bd ± 0.11	8.92Bd ± 0.06	8.73Bc ± 0.18
	F4	44.82Ac ± 0.82	41.70Bc ± 0.30	38.13Cc ± 0.36	35.27Dc ± 0.58	31.18Eb ± 1.09
	F5	46.14Ab ± 1.14	44.09Bb ± 0.09	40.10Cb ± 0.86	38.19Db ± 0.12	36.83 Da ± 0.15
	F6	50.80Aa ± 0.20	44.89Ba ± 0.09	42.17Ca ± 0.48	39.16 Da ± 0.09	37.19Ea ± 1.01
Yellow flavonoid	F1	1.63Ac ± 0.02	1.62Ae ± 0.06	1.60Ac ± 0.03	1.58Ac ± 0.01	1.52Ae ± 0.05
	F2	1.74Ac ± 0.04	1.72ABde ± 0.06	1.64ABc ± 0.03	1.61ABc ± 0.06	1.57Be ± 0.10
	F3	1.94Ac ± 0.04	1.93Ad ± 0.06	1.90Ac ± 0.20	1.87A c ± 0.02	1.84Ad ± 0.05
	F4	4.70Ab ± 0.90	4.33Ac ± 0.01	4.69Ab ± 0.63	4.61Ab ± 0.01	4.60Ac ± 0.01
	F5	5.24Ab ± 0.74	5.12Ab ± 0.07	5.10Ab ± 0.90	5.07Ab ± 0.17	5.02Ab ± 0.00
	F6	6.95Aa ± 0.85	6.83Aa ± 0.22	6.86Aa ± 0.17	6.78 Aa ± 0.55	6.41Aa ± 0.09
Ascorbic Acid	F1	9.87Ae ± 0.36	8.79Be ± 0.01	8.35BCe ± 0.06	8.21Ce ± 0.19	7.94Ce ± 0.15
	F2	10.63Ade ± 0.31	9.47Be ± 0.10	9.34Bde ± 0.08	8.73Ce ± 0.09	7.81De ± 0.22
	F3	12.23Ad ± 0.02	11.30Bd ± 0.20	10.18Cd ± 0.02	9.85Dd ± 0.01	9.81Dd ± 0.03
	F4	25.10Ac ± 0.09	24.30B c ± 0.10	23.47Cc ± 0.01	22.19Dc ± 0.00	22.07Dc ± 0.01
	F5	33.40Ab ± 0.35	32.09Ab ± 1.00	32.83ABb ± 0.90	30.57BCb ± 0.52	30.02Cb ± 0.02
	F6	41.68Aa ± 1.36	40.23ABa ± 0.2	39.27Ba ± 0.22	38.70BCa ± 0.30	37.25ca ± 0.15

The results are expressed as follows: *mg GAE 100 g⁻¹; ^** mg ECG 100 g⁻¹; ^*** mg 100 g⁻¹. F1, F2 and F3 (dairy beverage with 12%, 15% and 18% frozen red jambo pulp addition); F4, F5 and F6 (dairy beverage with 3%, 6% and 9% lyophilized red jambo pulp addition). Different capital letters on the same line indicate significant differences between the storage periods of the drinks. Different lowercase letters in the same column indicate significant differences between formulations (P < 0.05). Mean ± standard deviation, n = 3

The highest content of phenolic compounds was observed in formulations F3 (47.63 mg GAE 100 g⁻¹) and F6 (72.08 mg GAE 100 g⁻¹). However, there was a significant reduction in these values during 28 days of storage (Table 4). Likewise, Karaaslan et al. (2011) also reported a reduction in the content of phenolic compounds in yogurt supplemented with grape extract over the storage time. This effect may be related to the decomposition of polymeric phenolic compounds in the presence of lactic acid bacteria during cold storage (Cho et al. 2017) also to the interaction between these compounds with milk proteins, forming insoluble complexes (Raikos et al. 2019).

The total anthocyanin content also decreased during the storage period and differed significantly between all formulations evaluated (Table 4). Similarly, Silva et al. (2017) obtained a reduction in the total anthocyanin content of grape probiotic goat yogurt during 28 days of storage. This effect was already expected, since anthocyanins are highly reactive pigments, dependent on factors such as pH, enzymatic activity and microbial action and can degrade quickly during storage (Raikos et al. 2019). In addition, in milk-based products, these compounds can form complexes with macromolecules, reducing their availability (Geraldi et al. 2018).

The levels of yellow flavonoids, except for formulation F2, were not significantly affected during storage and were the lowest among all bioactive compounds evaluated. Such low results obtained were also reported in yogurt with Sulla honey (Perna et al.2014). Additionally, the beverages showed a significant reduction in the ascorbic acid content during the storage period (Table 4). It is known that ascorbic acid is highly unstable resulting in difficult incorporation and maintenance in different food systems (Abbas et al. 2012). Despite the ascorbic acid content reduction, the results were higher than those observed in a previous study with cherry yogurt (Vosgan et al.2016).

Formulation F6 with 9% lyophilized jambo pulp addition showed significantly higher antioxidant activity (16.21 μmol TE g⁻¹) than F3, with 18% frozen jambo pulp addition (12.10 μmol TE g⁻¹), consistent with the contents of bioactive compounds presented. However, the results of both are significant when compared to the antioxidant activity of some tropical fruit (Rufino et al. 2010) as well as when compared to the lack of antioxidant activity of probiotic dairy beverages without the addition of pulp (Cheuczuk et al. 2018).

Probiotic viability, starter cultures counts and microbiological analysis

Coliforms and Salmonella sp. were not detected, indicating how efficient the pasteurization process was and how good manufacturing practices were used to produce the dairy beverages.

Figure 2A shows the viable cell counts of the probiotic Lacticaseibacillus paracasei during refrigerated storage. Probiotic counts ranged from 8.23 to 7.85 log CFU mL⁻¹ and from 8.58 to 7.38 log CFU mL⁻¹, respectively in the formulations with the addition of frozen pulp and with lyophilized pulp during 28 days of storage. This indicated a significant probiotic reduction over time for all tested formulations, justifiable by the lowering of the pH values and a concomitant increase in total acidity during storage (Kumar and Kumar 2016). However, despite this reduction, the probiotic culture counts remained within the recommended minimum limit that varies between 7 to 8 log CFU mL⁻¹ in the final product (Ranadheera et al. 2019).

Fig. 2.

Header: Viability of Lacticaseibacillus paracasei (A), Lb. delbrueckii subsp. bulgaricus (B) and S. thermophilus (C) in lactose-free probiotic fermented goat dairy beverage formulations with red jambo pulp addition during 28 days of refrigerated storage. Footer: F0 (Control), F1, F2 and F3 (dairy beverage with 12%, 15% and 18% frozen red jambo pulp); F4, F5 and F6 (dairy beverage with 3%, 6% and 9% lyophilized red jambo pulp). Different capital letters at different time intervals indicate significant differences between storage periods for each beverage. Different lowercase letters in the same period indicate significant differences between formulations (P < 0.05). Mean ± standard deviation, n = 3

These probiotic count results differ from the ones reported by Pimentel et al. (2012), who found a significant increase in the viability of L. casei 01 during the same storage period in low-fat yogurt made with bovine milk, and were close to the findings of Andrade et al. (2019), who found a reduction in the viability of Lactobacillus acidophilus La-5 in a dairy beverage with the addition of papaya and orange pulp over time, demonstrating that maintenance of the probiotic strain viability varies according to the type of matrix and the culture used (Ranadheera et al. 2012).

Importantly, the addition of jambo pulp did not have a negative influence on the viability of the probiotic culture or the starter cultures (Fig. 2A, 2B, 2C), considering that the control sample (F0) did not present higher counts than the flavored formulations. Kumar and Kumar (2016) also did not observe any significant influence of the addition of the pulp on the viability of the probiotic culture when it is in probiotic yogurt made with bovine milk and jambolan pulp.

The initial viability of S. thermophilus did not differ significantly among the seven formulations, ranging from 10.53 to 10.34 log CFU mL⁻¹, unlike Lb. delbrueckii subsp. bulgaricus (7.58 to 7.04 log CFU mL⁻¹); however, a significant reduction over the storage time was recorded for both strains and corroborated the findings of Buriti et al. (2014), who also observed a significant reduction in the viability of S. thermophilus (T-40) in a probiotic goat milk beverage with the addition of guava pulp over 21 days. The cell populations of Lb. delbrueckii subsp. bulgaricus were lower than those of S. thermophilus, ranging from 7.21 to 6.84 log CFU mL⁻¹ in the beverages with the addition of frozen pulp and from 7.58 to 6.38 log CFU mL⁻¹ in those with lyophilized pulp addition, which may be related to greater sensitivity to oxygen than S. thermophilus (Silva et al. 2017). However, low counts of Lb. delbrueckii subsp. bulgaricus are more interesting, since this culture is the main culture responsible for reducing the pH of fermented products during storage, affecting the probiotic viability (Pimentel et al. 2012).

Sensory analysis

Color, aroma, and flavor attributes differed between the evaluated beverage formulations and over the storage period (Table 5). The reduction in the average scores of the color attribute over time are associated to the discoloration related to the degradation of anthocyanins present in the pulps, while the reductions in flavor and aroma attributes were probably due to the increase in acidity of the products during storage. These results are supported by Silva et al. (2017), who reported a reduction in the averages of the same attributes when evaluating probiotic goat yogurt with the addition of a grape preparation at 15%, 20% and 25% during 28 days of storage.

Table 5.

Values of the color, aroma, flavor attributes and global acceptance of lactose-free probiotic fermented goat dairy beverage formulations with red jambo pulp (frozen and lyophilized) addition during 28 days of refrigerated storage

		1 day	7 days	14 days	21 days	28 days
Color	F1	7.74Ad ± 1.22	7.67Ac ± 1.59	7.19ABbc ± 0.63	6.84Bb ± 1.06	6.67Bbc ± 0.81
	F2	8.30Abc ± 0.68	7.84Bbc ± 1.03	7.42BCb ± 0.90	7.03CDb ± 0.75	6.81Dabc ± 0.72
	F3	8.89Aa ± 0.31	8.46Ba ± 0.60	8.10Ca ± 0.57	7.58 Da ± 0.70	7.14Ea ± 0.66
	F4	7.98Acd ± 1.08	7.38Bc ± 1.14	7.03BCbc ± 0.84	6.82Cb ± 0.78	6.63Cc ± 0.73
	F5	8.44Ab ± 0.59	7.58Bc ± 1.44	6.89Cc ± 0.65	7.01Cb ± 1.14	6.93Cabc ± 0.94
	F6	8.96Aa ± 0.18	8.38 Bab ± 0.65	7.30Cb ± 0.80	7.16Cab ± 0.90	7.07Cab ± 0.70
Aroma	F1	7.44Ab ± 1.25	7.24Ab ± 1.30	6.98ABc ± 0.72	6.61Bab ± 0.99	6.56Bbc ± 0.63
	F2	7.79Aab ± 0.94	7.51Aab ± 1.36	7.35Aabc ± 0.77	6.72ABab ± 1.30	6.70Babc ± 0.88
	F3	7.84Aab ± 1.01	7.73Aa ± 0.93	736ABabc ± 1.03	7.16Ba ± 1.24	7.07Ba ± 1.25
	F4	7.53Aab ± 0.86	7.35Ab ± 1.20	7.09Abc ± 0.43	6.47Bb ± 1.02	6.44Bb ± 0.71
	F5	7.81Aab ± 0.93	7.61Aab ± 1.33	7.37ABab ± 0.64	6.95Bab ± 1.09	6.91Babc ± 0.95
	F6	8.02Aa ± 0.69	7.98Aa ± 0.83	7.53Ba ± 0.50	7.05Ca ± 0.61	7.01Cab ± 0.84
Flavor	F1	7.47Ab ± 1.67	7.35ABc ± 0.89	6.95ABCb ± 0.77	6.84BCb ± 0.70	6.51Cab ± 0.85
	F2	7.91Aab ± 1.07	7.51ABbc ± 0.68	7.28Bab ± 0.94	7.07Bab ± 0.73	6.56Cab ± 1.00
	F3	8.24Aa ± 0.95	7.95ABa ± 0.99	7.49BCa ± 0.83	7.31CDa ± 0.83	7.01 Da ± 0.90
	F4	7.88Aab ± 1.00	7.72ABabc ± 0.95	7.26Bab ± 0.94	6.75Cb ± 0.76	6.26Db ± 1.04
	F5	7.93Aab ± 0.86	7.84Aab ± 0.62	7.15Bab ± 1.11	6.91Bb ± 0.66	6.89Bab ± 1.02
	F6	8.23Aa ± 0.98	7.88Aab ± 0.57	7.17Bab ± 0.85	7.09Bab ± 0.71	6.95Ba ± 1.06
Consistency	F1	7.70Aa ± 0.81	7.44ABa ± 0.69	7.35 ABa ± 0.74	7.30 ABa ± 0.59	7.10 Ba ± 0.83
	F2	7.54 Aa ± 0.78	7.33 ABa ± 1.02	7.17 ABa ± 0.91	7.14 ABa ± 0.19	7.03 Ba ± 0.34
	F3	7.51 Aa ± 0.93	7.28 ABa ± 0.64	7.14 ABa ± 0.87	7.10 ABa ± 0.72	7.02 Ba ± 0.66
	F4	7.60 Aa ± 0.54	7.49 ABa ± 0.90	7.35 ABa ± 0.91	7.19 ABa ± 0.15	7.07 Ba ± 0.24
	F5	7.72 Aa ± 0.19	7.51 ABa ± 0.56	7.42 ABa ± 1.07	7.33 ABa ± 0.47	7.17 Ca ± 0.30
	F6	7.80 Aa ± 0.35	7.60 ABa ± 1.32	7.56 ABa ± 0.93	7.47 ABa ± 0.56	7.18 Ca ± 1.10
Global acceptance	F1	7.70Ac ± 1.12	7.36ABc ± 0.77	7.17Bb ± 0.19	7.02Bb ± 0.59	6.53Cb ± 0.25
	F2	7.86Abc ± 0.87	7.65ABbc ± 0.89	7.33BCb ± 0.27	7.16CDab ± 0.82	6.74Dab ± 0.38
	F3	8.53Aa ± 0.64	8.26ABa ± 1.17	8.10Ba ± 0.54	7.58Ca ± 0.31	7.23Ca ± 0.62
	F4	7.77Ac ± 0.73	7.45ABc ± 0.44	7.21ABb ± 0.67	7.10Cab ± 0.28	6.61Db ± 0.74
	F5	7.91Abc ± 0.52	7.47ABc ± 0.29	7.31Bb ± 0.93	7.12BCab ± 0.44	6.70Cab ± 0.29
	F6	8.35Aab ± 0.95	7.92Aab ± 0.69	7.40Bab ± 1.01	7.32Bab ± 0.91	7.01Bab ± 0.73

F1, F2 and F3 (dairy beverage addition of 12%, 15% and 18% frozen red jambo pulp addition); F4, F5 and F6 (dairy beverage with 3%, 6% and 9% lyophilized red jambo pulp addition). Different capital letters at different time intervals indicate significant differences between storage periods for each beverage. Different lowercase letters in the same period indicate significant differences between formulations (P < 0.05). Mean ± standard deviation, n = 3

The highest initial averages among the attributes corresponded to color, and the best scores were for formulations with a higher concentration of frozen (F3) and lyophilized (F6) pulp. Flavor and aroma also showed increased scores due to the increase in jambo pulp concentration. Thus, the addition of the pulps resulted in a more attractive beverage color and the most pleasant flavor and aroma attributes, positively influencing acceptance. Regarding the consistency attribute (Table 5), the averages differed significantly over storage time but not between the evaluated formulations. Accordingly, Buriti et al. (2014) also found such behavior for the same attributes in a goat probiotic milk beverage with 15% guava pulp.

The global acceptance decreased significantly over the storage time and differed between the evaluated formulations. Beverages were considered accepted when they presented global acceptance means ≥ 7.0 (Costa et al. 2016). Following this criterion, all formulations showed satisfactory acceptance during the 21 days of storage; however, formulation F3 stood out from the others during the entire evaluation period, with scores ranging from 8.5 to 7.2 (Table 5). Acceptance averages lower than those obtained in this research were recorded by Ranadheera et al. (2012) in goat probiotic yogurt, both natural and with mixed fruit juice after one week of storage.

Regarding purchase intention, the beverages differed depending on the storage period and between the evaluated formulations (Table S1). F3 formulation presented higher scores for 28 days, corresponding to the hedonic terms "always buy" and "buy often", which confirm the good acceptance of this formulation and point to its consumption prospects. The other formulations reached lower averages, consistent with those reported by Silva et al. (2017), in goat probiotic yogurt added with grape preparation, for 28 days.

Multivariate analysis

The first four main components contributed to explaining 75.67% of the total variation of bioactive compounds and sensory parameters of the formulations (Fig. 3A), also PC1 (39.24%), PC2 (14.99%), PC3 (10.97%) and PC4 (10.47%) were considered sufficient to represent the differences between samples on the 1st day of refrigerated storage. Four of the analyzed variables were the most important for the total variation in the first component (PC1): phenolic (0.97), anthocyanins (0.98), flavonoids (0.99) and ascorbic acid (0.99), as the correlation between these variables (load factor) was above 0.70 (Cruz and Regazzi 2003). Purchasing intention and aroma were the most important sensory variables to explain variations in PC3 (10.97%) and PC4 (10.47%), respectively.

Fig. 3.

Header: Principal component analysis (PCA) of bioactive compounds and sensory attributes in lactose-free probiotic fermented goat dairy beverage formulations with red jambo pulp addition after 1 (one) day (A) and 28 days (B) of refrigerated storage

The first four main components contributed to explaining 71.97% of the variation in the data after 28 days of storage (Fig. 3B), which was lower than the value found on the first day, confirming that there were changes in the concentrations of bioactive compounds and in the responses to sensory attributes of the beverages over storage time. The data variation was distributed between PC1 (37.37%), PC2 (13.32%), PC3 (11.24%) and PC4 (10.0%), and the variables belonging to the bioactive compounds with greater importance for the total variation were also grouped in PC1: phenolic (0.89), anthocyanins (0.98), flavonoids (0.99) and ascorbic acid (0.99), while the most important variables for the variation of PC2 were flavor (0.74) and PC3, global acceptance (0.73), indicating changes in attributes related to the sensory quality of the beverages.

Therefore, in this study, during the beverage storage period, PC1, which was associated with bioactive compounds, stood out as more important for the quality of the beverages than the other main components related to sensory attributes. Thus, based on the PCA, it is possible to consider the higher content of bioactive compounds as a sufficient parameter to confirm that F6 was superior to F3, although the latter had higher global acceptance and purchase intention scores.

Conclusions

The goat milk beverage formulations with jambo pulp addition showed relevant technological characteristics and stood out, especially as a source of bioactive compounds and antioxidant activity. Moreover, the addition of red jambo pulp in the beverage formulations did not negatively influence the viability of Lacticaseibacillus paracasei; therefore, it can be considered a propitious matrix for the maintenance of satisfactory viability of this probiotic culture. Finally, formulation F6, with the addition of 9% w/v lyophilized pulp, is the formulation whose concentration and type of pulp are most appropriate for obtaining a lactose-free goat probiotic fermented milk with improved bioactive properties targeting consumers who are lactose intolerant and consumers who are allergic to bovine milk proteins.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

GAE

Gallic acid equivalent

cyd-3-glu

Cyanidin-3-glycoside

TE

Trolox equivalent

Authors’ contributions

NGA—conception and design of study; acquisition of data; analysis and/or interpretation of data; drafting the manuscript; revising the manuscript critically for important intellectual content; approval of the version of the manuscript to be published. IMB – acquisition of data; approval of the version of the manuscript to be published. TLSL—analysis and/or interpretation of data; approval of the version of the manuscript to be published. RTM—drafting the manuscript; approval of the version of the manuscript to be published. HRC—conception and design of study; acquisition of data; analysis and/or interpretation of data; drafting the manuscript; revising the manuscript critically for important intellectual content; approval of the version of the manuscript to be published.

Funding

Not applicable.

Code availability

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Conflicts of interest

The authors declare they have no conflicts of interest or competing interests.

Ethics approval

This research was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (2.063.798/CAAE: 65937117.3.0000.5537).

Consent to participate

Not applicable.

Consent for publication

Not applicable.