Production of Kefir Powdered Milk Beverage Based on Probiotic Bacteria Enriched with Lupin, Kiwicha, and Quinoa ^†

Abstract

The production of functional foods has aroused growing interest due to its proven health benefits and potential to improve quality of life. One of the products that have gained importance due to its practicality is enriched beverages. Kefir, a fermented beverage traditionally produced from cow’s milk, is cultivated using kefir grains containing a symbiotic culture of bacteria and yeast, which has great nutritional power and benefits the microbiota. In this research, an enriched powdered milk beverage with a high protein content is prepared due to the incorporation of lupine (Lupinus mutabilis), kiwicha (Amaranthus caudatus), and quinoa (Chenopodium quinoa). The beverage prepared shows a 48% increase in protein content compared to commercialized kefir. After the beverage was obtained, it was freeze-dried to preserve its nutritional and functional properties. The resulting beverage, kefir milk powder, enriched with native Andean grains such as lupin, kiwicha, and quinoa, presents a sustainable and nutrient-rich option that contributes to dietary diversification.

1. Introduction

The development of novel functional and probiotic beverages is a growing trend in the food industry, driven by the increasing demand for healthier products [1]. One of the most widely consumed foods globally is dairy products, which are beneficial for health owing to their calcium content; kefir, a fermented dairy drink, is particularly beneficial for gut health [2]. Its nutritional profile can be further enhanced by adding Andean grains such as lupin, kiwicha, and quinoa, which are well-known for their high protein content [3]. Previous research has employed lupin to enhance the nutritional value of fresh cow’s milk, improving its protein content by 31.03% [4]. Mendoza [5] prepared a functional beverage based on quinoa and pineapple, rich in minerals and proteins, which exhibited a 6.4-fold increase in protein content compared to a conventional pineapple beverage. Quinoa (Chenopodium quinoa), kiwicha (Amaranthus caudatus), and lupin (Lupinus mutabilis) are known for their high nutritional value, including lipids, proteins, dietary fiber, and a wide range of minerals. Therefore, the combination of kefir and these legumes can result in a highly nutritious product. This research focuses on the production of a functional probiotic beverage based on kefir, a promising source of proteins and nutrients, using quinoa, kiwicha, and lupin.

2. Materials and Methods

2.1. Preparation and Fermentation of Kefir

One liter of whole cow’s milk was preheated to 25 °C and transferred to a glass container. Twenty grams of kefir grains, a consortium of acetic and lactic acid bacteria (LAB) and yeasts encompassing 20 different strains, were added. The flask was covered with sterile muslin cloth and incubated at 25 °C in the dark. After 24 h, the liquid was separated from the kefir grains using a sterile plastic sieve. Kefir grains that passed through the sieve were washed with sterile water and returned to fresh whole milk. All ingredients were purchased from a local market in Lima, Peru.

2.2. Enrichment with Lupin, Kiwicha and Quinoa

Lupin grains were soaked for 12 h to remove bitterness. Subsequently, 150 g of lupin grains, 30 g of kiwicha flour, and 70 g of quinoa flour were added to 700 mL of kefir milk. The mixture was blended and pasteurized at 90 °C for 15 min. All ingredients were purchased from a market in Lima, Peru.

2.3. Freeze-Drying of the Final Product

The sample was subjected to a vacuum sublimation process at −50 °C for 24 h in a freeze-dryer (Martin Christ Alpha 1–2, Zirbus VaCo 5) to obtain the enriched kefir drink powder. The powder obtained (EKM) was stored in sealed polyethylene bags under vacuum conditions for quality analysis.

2.4. Proximal Composition

Proximate composition was determined according to official methods. Moisture content was determined at 110 °C to constant weight. Ash content was determined by incineration at 550 °C for 72 h in a muffle furnace. Total protein content was determined using a Kjeldahl analyzer (UDK 139, VELP, Usmate Velate, Monza, Italy). Fat content was determined using n-hexane extraction for nine hours. All measurements were performed in triplicate [6].

2.5. Determination of Mineral Content

The concentrations of Ca, Cu, Fe, K, Mg, Na, P, and Zn in the powdered beverage were determined by atomic absorption spectrophotometry (Nexion 350×, Perkin Elmer, MA, USA) [6].

2.6. Physical-Chemical Properties

Apparent and compacted densities, Hausner ratio, and solubility were determined according to the methodologies outlined by Chasquibol et al. [6]. Hygroscopicity was determined according to the methodology described by Nowshina et al. [7]. A Brookfield rotational rheometer (model DV-II Pro LV; Brookfield Engineering Laboratories, Middleboro, MA, USA) was used for viscosity analysis. The data obtained were processed using the Rheocalc application software (V3.2 Build 47-0; Brookfield Engineering Laboratories, Middleboro, MA, USA) according to the methodology described by Kok-Tas et al. [8].

2.7. Color Measurements

Color measurements were performed using a Color Tec-PCM colorimeter (Model D25-PC2, Cole Palmer, Vernon Hills, Chicago, IL, USA) according to the methodology described by Nowshina et al. [7].

2.8. Amino Acid Determination

The amino acid profile was quantified by HPLC (ARC, Waters, Milford, CT, USA) following the methodology outlined by Chasquibol et al. [6]. All measurements were performed in triplicate.

2.9. Sensory Analysis

Fifteen semi-trained panelists evaluated the sensory analysis (color, flavor, aroma, texture, and overall acceptability) of the powder following the methodology presented by Nowshina et al. [7].

2.10. Statistical Analysis

Results were expressed as mean ± standard deviation. All measurements were performed in duplicate or triplicate. Analysis of variance (ANOVA) was used for the mixture design employed in this study and for response optimization of the protein variable. Data were analyzed at a 95% significance level using Minitab 19.0 statistical software (Minitab Inc., State College, Palo Alto, PA, USA).

3. Results and Discussion

3.1. Statistical Analysis

A simplex centroid mixture design was used to optimize the protein content in a formulation composed of kefir milk enriched with lupin, kiwicha, and quinoa, as depicted in

Table 1. Statistical Analysis.

RunOrder	PtType	Blocks	Kefir Milk	Lupin	Proteins Content
1	−1	1	0.725	0.125	32.27
2	1	1	0.700	0.150	37.92
3	−1	1	0.775	0.075	19.29
4	0	1	0.750	0.100	23.86
5	1	1	0.800	0.50	14.70

. The design consisted of five experimental runs, varying the levels of kefir milk (700–800 mL) and lupin (50–150 g). The results indicated that the maximum protein content was achieved with a mixture containing 700 mL of kefir milk and 150 g of lupin, as depicted in Figure 1. The quantities of kiwicha and quinoa were held constant throughout the experiment.

3.2. Proximal Composition and Mineral Content, Recommended Dietary Intake (RDI), and Upper Tolerance Limit (UTL)

The Andean grains proved to be highly effective in augmenting the protein content of the product, as evidenced by the data presented in

Table 2. Proximal composition, mineral content, recommended dietary intake (RDI), and upper tolerance limit (UTL).

Proximal Composition (%)	EKM (g/100 g)	Minerals	EKM (mg/kg)	RDI * (mg/day)	UTL (mg/day)
Moisture	0.34 ± 0.002	Calcium	2505.50 ± 4.02	1000	2500
Ash	3.59 ± 0.02	Copper	4.20 ± 0.02	0.9	8
Protein	37.92 ± 0.02	Iron	42.30 ± 0.10	W 18, M 8	45
Fat	18.82 ± 0.02	Potassium	5877.11 ± 7.04	W 2600, M 3400	3400
		Magnesium	1375.96 ± 3.07	W 315, M 410	350
		Sodium	1644.69 ± 2.01	2000	2300
		Phosphorus	4799.81 ± 3.02	700	4000
		Zinc	31.53 ± 0.07	W 8, M 11	40

Values are mean ± SD; n = 3. * For women (W) and men (M), aged 19 to 50 years.

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The protein content increased from 14.70 g to 37.92 g. Furthermore, the moisture content was measured to be 0.34 ± 0.002 g per 100 g. The freeze-drying method can eliminate all the liquid content of the sample and the storage of the product has to be in sealed polyethylene bags under vacuum conditions to prevent an increase in moisture, bacterial activity, and the degradation of the product. The determination of the ash content of the EKM showed a 3.59 ± 0.02 g/100 g product.

EKM contains a wide range of minerals, as indicated in Table 2; if it is compared with the commended daily intake for each mineral (National Institute of Health, 2023) [6], the consumption of only 20 g daily of EKM would cover 5.01% of the daily requirement of calcium, 12.29% of iron, 4.52% of potassium, 8.73% of magnesium, and 13.71% of phosphorus. Therefore, EKM offers a nutritious option full of various minerals that contribute to a balanced diet.

3.3. Physical-Chemical Analyses

Table 3. Physical-chemical properties of the product (EMK).

Analysis	EKM
Solubility %	26.96% ± 0.002
Hygroscopicity (g)	0.03 ± 0.002
Water activity	0.86 ± 0.001
Bulk density (g/cm3)	6.9 ± 0.001
Tapped density (g/cm3)	4.2 ± 0.003
L*	78.1 ± 1.2
a*	−1.59 ± 0.02
b*	4.89 ± 0.02

Values are mean ± SD; n = 3.

shows physical-chemical analyses of the product (EMK). The solubility value of EKM obtained was 26.96% ± 0.02 due to the higher fiber content of lupin, kiwicha, and quinoa. The value for hygroscopicity was 0.03 ± 0.02, indicating that the EKM sample has a lower capacity to absorb moisture from the environment, implying reduced absorption of vapor or liquid. This low moisture content in powdered milk inhibits the growth and reproduction of microorganisms. Since hygroscopic ingredients are sensitive to moisture, and in powdered milk, it can lead to deterioration, such as softening, color changes, and disintegration [2]. Therefore, the water activity in EKM was e 0.86 ± 0.01, which agrees with the aforementioned findings. By maintaining low moisture content, the proliferation of bacteria, molds, and yeasts is effectively controlled. The EKM exhibited an apparent density of 6.9 ± 0.01 g/mL and a compacted density of 4.2 ± 0.03 g/mL, with a Hausner ratio 0.6. It also possessed a viscosity of 20.06 mPa·s, indicating optimal fluidity. These values suggest that the powdered beverage has good flowability.

For the color analysis of EKM, the L*, a*, and b* values of the samples were analyzed after preparation according to the formula. The EKM shows a higher value of luminosity (L *); this is because it is made from cow’s milk, and it retains a color close to chalk white, which is quite similar to quinoa and kiwicha flour; the lupin gives it a yellowish cream appearance; that gives color to EKM a color is obtained in the range of white, lead and cream, a color close to ivory, which matched to milk measurements. The a* represents the redness or greenness of the products; in EKM, a negative value was shown, which represents the greenness. The b* value represents yellowness values or a blue indicator; in EKM, a positive value is shown, which represents yellowness in the sample. The low values of greenness and yellowness are due to the original color of the lupin in EKM [7].

3.4. Amino Acids in the Product

Table 4. Amino acid profile of the product (mg/g protein).

Aminoacid	EKM
Aspartic Acid	37.61 ± 0.04
Glutamic Acid	85.80 ± 0.26
Serine	21.55 ± 0.08
Histidine	8.31 ± 0.22
Glycine	18.61 ± 0.43
Threonine	16.09 ± 0.47
Arginine	46.14 ± 0.14
Alanine	13.93 ± 0.06
Proline	25.24 ± 0.43
Tyrosine	14.02 ± 0.35
NH3	27.93 ± 0.30
Valine	21.55 ± 0.35
Methionine	7.62 ± 0.22
Cystine	1.58 ± 0.01
Isoleucine	17.28 ± 0.45
Leucine	39.63 ± 0.22
Phenylalanine	19.09 ± 0.12
Lysine	21.10 ± 0.36
Tryptophan	4.05 ± 0.55

Values are mean ± SD; n = 3.

presents the results of the amino acid profile of the product, a sample of kefir milk powder enriched with lupin, kiwicha, and quinoa (EKM). The protein properties of lupin, kiwicha, and quinoa have benefited the EKM by allowing them to retain better concentrations of essential amino acids. Essential amino acids cannot be synthesized within the body and, therefore, must be provided through the diet [9]. The predominant amino acids in EKM are glutamic acid (85.5 ± 0.26 mg/g of protein), arginine (46.14 ± 0.14 mg/g of protein), and leucine (39.63 ± 0.22 mg/g of protein), which is, according to informed by Akal [9], aspartic acid (37.61 ± 0.04 mg/g protein), NH 2 (27.93 ± 0.30 mg/g protein), and proline (25.24 ± 0.43 mg/g protein); the limiting amino acids are methionine 7.62 ± 0.22 mg/g protein and tryptophan 4.05 ± 0.55 mg/g protein. The amino acid composition of EKM showed a balanced profile in accordance with FAO/WHO recommendations and demonstrated that dairy drinks formulated with lupin, kiwicha, and quinoa are rich in amino acids, except for tryptophan [7].

3.5. Sensory Evaluation

Table 5. Average score of overall acceptability of the product (EKM).

Formulation	EKM
Color	7.1 ± 0.02
Flavor	6.7 ± 0.02
Arome	7.9 ± 0.02
Texture	7.3 ± 0.02
Overall Acceptability	7.2 ± 0.03

shows the results of 15 panelist’s evaluation of the organoleptic characteristics for EKM.

EKM obtained a score of 7.1 for color, indicating a similar color to commercial milk. This is in contrast to the color indicator reported by Nowshina et al. [7], which ranged from 5.3 to 6.7 due to the addition of maltodextrin and sunflower oil, causing a color change from yellow to brown, and was not well-accepted by their panelists. Regarding flavor, EKM scored 6.7, as it maintained a neutral taste (similar to almond milk, according to four panelists). EKM received a score of 7.9 for aroma, characterized by a unique flavor profile typical of kefir milk, with a predominant aroma of quinoa and kiwicha, which were highly appreciated by the panelists. The texture was rated 7.3, as it was described as a fluid and easy-to-drink beverage. EKM achieved an overall acceptability score of 7.2 (on a 1–9-point rating scale), aligning with the panelists’ perceptions of color, flavor, aroma, and texture.

4. Conclusions

The formulation developed of kefir milk powder enriched with lupin, kiwicha, and quinoa enabled obtaining a beverage with high protein content. The addition of these Andean grains enhanced the product’s mineral content, particularly calcium, which is essential in dairy beverages. Moreover, it provided a significant amount of essential amino acids. The powder exhibited acceptable solubility with low hygroscopicity, moisture content, and water activity, thereby preventing microbial growth and improving product quality and shelf life. Sensory evaluation revealed acceptable characteristics in terms of color, flavor, texture, and aroma, indicating a generally positive consumer acceptance. In conclusion, kefir milk powder enriched with lupin, kiwicha, and quinoa represents a nutritious option, providing a significant source of protein and probiotic benefits to improve gut health and overall nutrition.