Beer production potentiality of some non-Saccharomyces yeast obtained from a traditional beer starter emao

Nitesh Boro; Ashis Borah; Rajib L Sarma; Diganta Narzary

doi:10.1007/s42770-022-00765-7

. 2022 Apr 29;53(3):1515–1531. doi: 10.1007/s42770-022-00765-7

Beer production potentiality of some non-Saccharomyces yeast obtained from a traditional beer starter emao

Nitesh Boro ¹, Ashis Borah ¹, Rajib L Sarma ², Diganta Narzary ^1,^✉

PMCID: PMC9433491 PMID: 35488168

Abstract

The recent realisation regarding the potentiality of the long-neglected non-Saccharomyces yeasts in improving the flavour profile and functionality of alcoholic beverages has pushed researchers to search for such potent strains in many sources. We studied the fungal diversity and the rice beer production capability of the fungal strains isolated from emao—a traditional rice beer starter culture of the Boro community. Fifty distinct colonies were picked from mixed-culture plates, of which ten representative morphotypes were selected for species identification, and simultaneous saccharification and beer fermentation (SSBF) assay. The representative isolates were identified as Hyphopichia burtonii (Hbur-FI38, Hbur-FI44, Hbur-FI47 & Hbur-FI68), Saccharomyces cerevisiae (Scer-FI51), Wickerhamomyces anomalus (Wano-FI52), Candida carpophila (Ccar-FI53), Mucor circinelloides (Mcir-FI60), and Saccharomycopsis malanga (Smal-FI77 and Smal-FI84). The non-Saccharomyces yeast strains Hbur-FI38, Hbur-FI44, Ccar-FI53, and Smal-FI77 showed SSBF capacity on rice substrate producing beer that contained 7–10% (v/v) ethanol. A scaled-up fermentation assay was performed to assess the strain-wise fermentation behaviour in large-scale production. The nutritional, functional, and sensory qualities of the SSBF strain fermented beer were compared to the beer produced by emao. All the strains produced beer with reduced alcohol and energy value while compared to the traditional starter emao. Beer produced by both the strains of H. burtonii stood out with higher ascorbic acid, phenol, and antioxidant property, and improved sensory profile in addition to reduced alcohol and energy value. Such SSBF strains are advantageous over the non-SSBF S. cerevisiae strains as the former can be used for direct beer production from rice substrates.

Graphical abstract

graphic file with name 42770_2022_765_Figa_HTML.jpg

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-022-00765-7.

Keywords: Traditional beer starter, Functional beer, Non-Saccharomyces yeast, Hyphopichia burtonii, Candida carpophila, Saccharomycopsis malanga

Introduction

The origin of fermented alcoholic beverages goes far back in the history of human civilization. However, only during the nineteenth century, yeasts’ active role in alcoholic fermentation came into knowledge [1]. Saccharomyces cerevisiae is the common yeast frequently reported in spontaneous fermentation of various raw materials due to which it got rigorously selected over generations in various cultures. Qualities like high ethanol production, tolerance capability and preference for fermentation over respiration under high sugar concentrations of domesticated S. cerevisiae strains, eventually led to extensive use of this yeast for controlled fermentation in breweries all over the world [2]. In addition to Saccharomyces, a wide diversity of yeasts belonging to other genera has also been recorded from many sources like fruits [3–5], indigenous fermented foods, and beverages [6, 7].

Non-Saccharomyces yeasts generally dominate during the early stages of spontaneous fermentation [8] and play a vital role in determining the complexity in flavour and aroma of the final product [9]. Recent explorations have brought some of them into the spotlight as they have proved to be quite promising in bringing unique and desirable attributes to various alcoholic products even though they were earlier regarded as ‘spoilage yeasts’ [10]. Brettanomyces sp. and Torulaspora delbrueckii were reported to produce flavour precursors [11–13], and thus they are being used in some breweries to formulate alcoholic beverages with novel and complex aroma [2]. Moreover, the growing demands for specialised products like alcoholic beverages with health-promoting factors, low calorific value, and low alcohol content, which are the common functional beer attributes, also bring an immense number of possibilities that such non-conventional yeasts can render in brewing.

The diversity in culture and ethnicity of the people residing in northeast India provides a unique identity to the land. This diversity also extends to the types of fermented foods and beverages enjoyed by these ethnic groups, their ingredients, and the preparation method, most of which are still awaiting scientific exploration. The rice beer called ‘zu’ or ‘jou’ is prepared by the Boro (also written as ‘Bodo’) tribe of India using an amylolytic starter culture called ‘emao’ or ‘amao’ [14, 15], which is one such unexplored alcoholic beverage being prepared traditionally since time immemorial. Emao is prepared primarily from rice grain, certain herbs, and a small quantity of emao from a previous batch. The traditional method of emao and jou preparation by the Boro tribe has been described by Basumatary and Gogoi [16]. During emao and jou preparation, utmost care on hygiene is maintained as the quality deteriorates if not done carefully. Apart from being a refreshing drink, Boro people consider jou to be helpful in bowel and urinary disorders, insomnia, and tiredness after a day’s hard work. The use of numerous herbs of already-proven ethnomedicinal importance and the traditionally believed health-promoting properties of the drink escalate the possibility of discovering potential microbes from emao.

Even though some exploratory studies have been made on the methods and ingredients used to prepare such traditional rice beer starters of north eastern India, thorough attempts to understand the individual role of the fungal constituents on the quality and functionality of the beer product is still rare. Some non-Saccharomyces yeast has already been found useful to remove undesired derivatives in wine fermentation [17]. In the present study, we screened non-Saccharomyces yeasts associated with an age-old rice beer starter ‘emao’ of the Boro community for simultaneous saccharification and beer fermentation (SSBF) capability and compared their beer products to the traditional beer.

Materials and methods

Emao collection

A traditionally prepared 1-month-old emao sample was collected aseptically in a sterile sample container (Tarsons products, Kolkata) from a Boro village (Boragari, Dotoma, Assam, India). The sample was stored in a dry place at room temperature, and was processed for microbial isolation within a month of collection.

Isolation and preservation of the fungal strains

Isolation of fungi from emao was done on sterile Sabouraud dextrose agar (SDA) plates (40 g/L dextrose, 10 g/L mycological peptone, 15 g/L agar) supplemented with 25 µg/mL tetracycline using the spread-plate method. Morphologically distinct and well-differentiated colonies were sub-cultured onto fresh SDA plates and eventually purified onto SDA slants. The inoculated agar plates/slants were incubated at 25 ± 1 °C in a Biological Oxygen Demand (BOD) incubator.

The pure cultures were maintained in SDA slants at 4 °C for short-term storage and in sabouraud dextrose broth (SDB) medium (40 g/L dextrose, 10 g/L mycological peptone) supplemented with sterile glycerol (20% v/v) at − 80 °C (Eppendorf, Germany) for long-term storage. Pure culture isolates were again segregated according to their colony and micromorphological characters, and then representative isolates were considered for species identification and further SSBF assay.

Strain identification

The colony morphology of the isolates was recorded after 96 h of incubation at 25 ± 1 °C in SDA plates. Cellular morphology was studied under a trinocular compound microscope (Carl Zeiss, Germany) after staining with lactophenol cotton blue. Morphological and microscopic characters of the isolates were compared to species’ characteristics as depicted in the book of Kurtzman and Fell [18] for identification of the isolates.

Species identification was complemented and confirmed by the sequencing of the nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions and BLAST homology search of the sequences in the NCBI database. The genomic DNA was isolated following the method of Cenis [19]. The nrDNA ITS regions (including the 5.8S region) were amplified using the primer pairs: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [20] in a 25 µl reaction volume containing 0.4 μM of each primer, 200 μM each in equimolar ratios of dNTPs, 1 × Taq polymerase buffer A, 1 unit Taq DNA polymerase (Bangalore Genei, India), and ~ 50 ng genomic DNA. A Mastercycler GSX1 (Eppendorf, Germany) thermal cycler was programmed for 3 min pre-denaturation at 94 °C, then 35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 50 °C, and 1.5 min primer extension at 72 °C, and followed by a 5-min final extension at 72 °C and preservation at 4 °C [21]. The amplified products were separated by gel electrophoresis in 1% agarose gel using 1 × Tris–Acetate-EDTA buffer containing ethidium bromide and visualised in a UV transilluminator. The prominent DNA bands that appeared singly in respective wells were excised, extracted using QIAquick Gel Extraction Kit (QIAGEN), and sequenced by outsourcing. The obtained DNA sequences were deposited in the NCBI database.

Assessment of simultaneous saccharification and beer fermentation potential of the representative strains

Inoculum preparation

Representative fungal strains were inoculated individually into 60 mL of sterile SDB media and incubated in a shaking incubator (REMI, Mumbai) at 180 rpm at 27 ± 1 °C, keeping one uninoculated media as a negative control. After 9 days of incubation, the fungal biomass was harvested by centrifugation at 8000 × g for 10 min. The pellets were washed twice with sterile distilled water by centrifugation as above. Two grammes of each pure culture pellet was re-suspended in 25 mL sterile distilled water by brief vortexing and used immediately as inoculum for the SSBF assay on rice substrate. Accordingly, 2 g of emao suspended in 25 mL sterile distilled water was used as a positive control.

Preparation of rice substrate and inoculation

For SSBF assay of each representative isolate, 180 g of dehusked glutinous rice (local variety Maibra) was weighed, washed, and steamed in a 1-L capacity of borosilicate glass bottle (Borosil, Mumbai) by autoclaving at 15 lbs per square inch pressure for 15 min. The autoclaved bottles with rice were then taken out and allowed to cool down to about 35 °C inside a laminar airflow hood and were inoculated with their respective inoculum. A sterile glass rod was used to mix the contents thoroughly. The bottles were kept in the dark, dry place at room temperature (25 ± 2 °C) and were monitored weekly for 28 days. Liquefaction of rice substrate due to SSBF was considered as a positive result.

Scaled-up production with the SSBF-positive strains

The SSBF-positive fungal strains were further employed in a scaled-up rice beer production to understand their nutritional, nutraceutical, and sensory contributions in a full-scale brewing platform. For each strain, a 5-L capacity plastic jar with closing lid and a custom-made collection apparatus were used for fermentation (Graphical abstract, Step 9). All the apparatus were cleaned thoroughly with distilled water, air-dried, and sterilised using 70% ethanol and UV treatment (15 min) before use. Six kilogrammes of dehusked non-glutinous rice (local variety aijong) was baked in a stainless-steel pot as followed in the traditional method. Before cooling, the baked rice was divided into six equal amounts and each part (approx. 1 kg) was transferred into pre-sterilised plastic jars one by one. The inoculum was prepared and inoculated into the jars as described in the “Preparation of rice substrate and inoculation” section. After inoculation, the content of each jar was appropriately mixed and a custom-made collection apparatus was placed at the centre of each jar (Graphical abstract, step 9) to monitor beer formation and collect the beer for analysis. The jars were closed with their respective lids and were kept undisturbed in a dark and dry place at room temperature (25 ± 2 °C) for 28 days. Progress was monitored weekly, and beer samples were taken out using a sterile micropipette when the liquefaction was observed after 14, 21, and 28 days of inoculation for biochemical analysis.

Analyses of the beer samples

Initially, five parameters viz. pH, ethanol, total carbohydrates (TC), total reducing sugars (TRS), and free amino nitrogen (FAN) that play vital roles in growth physiology of yeast were estimated after 14, 21, and 28 days of strain inoculation. Due to the low volume of beer production at the earlier stages of fermentation, other twelve parameters viz. titratable acidity (TA), total phenol content, DPPH radical scavenging activity (SADPPH), reducing power (RP), total antioxidant activity (TAA), ascorbic acid, total soluble solids (TSSs), energy value (EV), free amino acids (FAAs), total protein (TP), colour, and sensory scores were estimated only after 28 days of brewing. All the estimations were done in triplicates (n = 3), except the sensory evaluation (n = 5), and the standard deviation (SD) was calculated for each parameter.

Determination of pH and total titratable acidity

The pH of each beer product was determined using a calibrated digital pH metre (Systronics, Mumbai) at room temperature (~ 25 °C). Standard buffer solutions of pH 4 and 7 were used for calibration of the pH metre.

TA was determined by titrating 10 mL of each sample with 0.1 N of sodium hydroxide solution containing phenolphthalein (1% w/v) until the phenolphthalein’s pink colour persisted for at least 15 s [22]. It was calculated using the following formula and was expressed in percent tartaric acid equivalent (%TAE) where the milliequivalent factor considered for tartaric acid was 0.075.

Titratable acidity = (Vol . of NaOH consumed \times 0.1 \times milliequivalent factor \times 100) / 10

Ethanol estimation

The concentration of ethanol in each beer product was determined by following a method described by Pai et al. [23] with minor modifications. One millilitre of diluted beer sample (1:20 v/v; with distilled water) was taken in a sample holder and placed suspended above 10 mL acid dichromate solution inside a conical flask (0.75 g of potassium dichromate in 70 mL concentrated sulphuric acid diluted to 250 mL with distilled water). The whole system was sealed using a rubber stopper and incubated at 35 ± 2 °C overnight. After that, 100 mL distilled water and 1 mL of 20% (w/v) potassium iodide solution were added to the acid dichromate and were titrated with 0.03 mol/L sodium thiosulphate solution using 1% starch solution (w/v) as indicator until the blue colour disappeared. Ethanol concentration was determined using the formula:

Ethanol (% w / v) = 0.03 \times (V_{b} - V_{s}) \times 0.25 \times dilution factor \times 46 / 10

where V_b is the volume of thiosulphate consumed for blank, and V_s is the volume of thiosulphate consumed for the sample.

The ethanol concentration was expressed in v/v after dividing the w/v concentration with ethanol’s specific gravity at 25 °C.

Determination of total carbohydrate and total reducing sugar

TC content was determined by following the method described by Dubois et al. [24] and using glucose as a standard. One millilitre of 5% (w/v) phenol solution was mixed with 2 mL of rice beer sample. To the mixture, 5 mL of sulphuric acid was added rapidly and allowed to stand for 10 min. The tube was then mixed thoroughly, and absorbance was taken at 490 nm using a UV–VIS spectrophotometer (Eppendorf, Germany) after colour development. A standard curve was prepared using glucose standards in the range of 0.02–0.1 mg/mL, and TC content was determined in mg/mL from the derived standard curve (Supplementary data S1).

TRS was estimated using 3,5-dinitrosalicylic acid (DNS) reagent [25]. Three millilitres of DNS reagent (containing 1 g DNS, 200 mg crystalline phenol, and 50 mg sodium sulphite in 100 mL 1% w/v sodium hydroxide) was added to 3 mL of rice beer sample and was heated in a boiling water bath for 15 min. The solution mixture was allowed to cool up to room temperature, and 1 mL of 40% (w/v) Rochelle salt solution was added to it. The absorbance of the mixture was taken at 510 nm. Known concentrations of glucose (0.1–0.5 mg/mL) were used to prepare the standard curve (Supplementary data S1). TRS value was expressed in g/L.

Total protein and free amino acids

Lowry’s method [26] was followed to determine the TP content. One hundred millilitres of solution A (prepared from 50 mL of 2% w/v sodium carbonate added to 50 mL 0.1 N NaOH solution) was mixed with 2 mL of solution B (prepared from 10 mL of 1.56% w/v copper sulphate solution mixed with 10 mL of 2.37% w/v sodium potassium tartrate solution) to prepare the alkaline copper solution which was then mixed with the beer sample in 1:1 ratio and kept for 10 min at room temperature. Then, 0.2 mL of 1 N folin-ciocalteu reagent was added and mixed. Absorbance was taken at 660 nm after 30 min. A standard curve prepared using bovine serum albumin standards (0.2–1 mg/mL) was used to calculate the TP of the beer samples (Supplementary data S1).

The FAA content was determined following a method described by Lee and Takahashi [27]. 0.2 mL of beer sample was added to 2 mL of ninhydrin-citrate buffer-glycerol solution and heated in a boiling water bath for 12 min. Ninhydrin-citrate buffer-glycerol solution was prepared mixing 1% w/v of ninhydrin solution (prepared in 0.5 M citrate buffer, pH 5.5) and 0.5 M citrate buffer (pH 5.5) and glycerol in 5:2:12 proportions, and the pH of the final solution was adjusted to 6 using citrate buffer. The solution was allowed to cool at room temperature and its absorbance was taken at 570 nm. FAA was calculated from a standard curve obtained from glycine standards (100–500 µg/mL) (Supplementary data S1) and it was expressed in g/L.

Free amino nitrogen

FAN was estimated by the photometric method of MEBAK (2017). Two millilitres each of 2% diluted beer sample (1:50, in distilled water), 1% standard glycine solution containing 2 mg/L of amino nitrogen, and a blank solution (distilled water) were taken separately in three different test tubes. Glycine stock solution (glycine stock: 107.2 mg glycine in 100 mL distilled water) was diluted with distilled water to make 1% standard glycine solution. To each tube, 1 mL of ninhydrin colour reagent (10 g sodium hydrogen phosphate, 6 g potassium dihydrogen phosphate, 0.5 g ninhydrin, 0.3 g fructose in 100 mL distilled water, and pH adjusted to 6.7) was added and adequately mixed. The tubes were then heated at 100 °C for 16 min and allowed to cool to 20 °C and stand for 20 min. Then, 5 mL of a diluent solution (2 g potassium iodide in 600 mL distilled water and 400 mL 96% ethanol) was added, and its absorbance was taken at 570 nm. FAN was calculated from the formula given below:

Free amino nitrogen = (A_{570} of the sample - A_{570} of blank) / (A_{570} of glycine standard - A_{570} blank) \times 2 \times Dilution factor of the sample

Total soluble solid

The TSS in the rice beer samples was determined using a calibrated hand refractometer (Erma, Japan) and it was expressed in the Brix unit.

Total phenol content

It was estimated following a method described by Singleton et al. [28]. A beer sample of 20 µL was diluted with 1.58 mL distilled water, and 100 µL of Folin-Ciocalteu reagents was added to it. The solution was mixed thoroughly, kept undisturbed for 5 min at room temperature, and then 300 µL of a saturated sodium carbonate solution was added to it. The absorbance of the solution was taken at 765 nm after 2 h of adding sodium carbonate. Similarly, a gallic acid standard curve was prepared using gallic acid solutions from 0.1 to 0.25 mg/mL (Supplementary data S1), and the phenol content of the samples was expressed in mg Gallic acid equivalent (GAE)/L.

Ascorbic acid

It was estimated by a method described by Costa et al. [29]. A beer sample of 25 mL was taken in an Erlenmeyer flask and titrated with 1% (w/v) iodine solution using 1% (w/v) starch solution as an indicator. The persistence of the blue-black colour of the iodine–starch compound indicated the end of the titration. A standard curve was prepared using ascorbic acid standards (from 0.2 to 1 mg/mL), and the ascorbic acid content of the samples was expressed in mg/100 mL.

Energy value

The EV of each beer sample was determined using MEBAK method as described by Olsovska et al. [30]. The EV value calculated in kJ/100 mL from the following formula was converted to kcal/100 mL using the standard (1 kcal = 4.1868 kJ).

EV = 17 \times C_{carbohydrate} + 17 \times C_{protien} + 29 \times C_{alcohol}

where alcohol, protein, and carbohydrate values are in g/100 mL.

DPPH radical scavenging activity

For the determination of the samples’ free radical scavenging activity, the method proposed by Chen et al. [31] was used. Ascorbic acid (0.2 mg/mL) was used as the positive control, and distilled water was used as the blank. Five millilitres of 0.1 mmol/L 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution in methanol was mixed with 5 mL of an aqueous solution containing 150 µL of a beer sample. After 30 min of incubation at room temperature in darkness, the absorbance was taken at 517 nm. The SADPPH was expressed as:

SADPPH (%) = (1 - Absorbance of sample solution / Absorbance of blank) \times 100

Reducing power (RP)

It was determined following the method of Oyaizu [32]. A beer sample of 150 µL was diluted with 850 µL of sterile water to make it 1 mL which was then mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% w/v potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. Again, 2.5 mL of 10% (w/v) trichloroacetic acid was added to it, and the mixture was centrifuged at 956 × g for 10 min. Then, 2.5 mL of the upper phase was taken out and mixed with 2.5 mL distilled water and 0.5 mL of 0.1% (w/v) ferric chloride in another fresh tube. The absorbance of the mixture was taken at 700 nm. Ascorbic acid solution (0.2 mg/mL) was used as a positive control. A higher absorbance at 700 nm indicates higher RP of the sample.

Total antioxidant activity (Phosphomolybdate assay)

Total antioxidant activity (TAA) was determined according to Prieto et al. [33] as described by Ahmed et al. [34]. A beer sample was diluted two times with sterile water of which 300 µL was taken for mixing with 3 mL of phosphomolybdate reagent (equal volume of 0.6 M sulphuric acid, 20 mM sodium phosphate, and 4 mM ammonium molybdate). The mixture was heated at 95 °C for 90 min, then cooled down to room temperature, and absorbance was taken at 765 nm. Ascorbic acid solution (0.2 mg/mL) was used as a positive control. The results were expressed in g ascorbic acid equivalent (AAE)/L.

Beer colour

It was determined by following the MEBAK method (2017). Beer samples were centrifuged at 4500 × g for 10 min, and then the absorbance of the supernatant was taken in a spectrophotometer at 430 nm. Beer colour was expressed in EBC value.

Sensory evaluation

Sensory evaluation of the rice beer samples was conducted with a panel of 5 members under ambient light and temperature following a questionnaire of the American Wine Society with some modifications (Supplementary data S2). All the members of the panel had earlier experience with traditional rice beer consumption. The panellists were randomly served with 20 mL of each sample in a transparent glass. Scores awarded for appearance, aroma, taste and texture, aftertaste, and overall impression were used to calculate each sample’s overall sensory score.

Statistical analyses

Standard deviation (s.d.) value was calculated for each parameter from triplicate (n = 3) measurements, except for the sensory evaluation (n = 5). Pearson’s correlation index and the analysis of variance (ANOVA) were performed at a statistical significance level (p < 0.05). Principal component analysis (PCA) biplot was generated by analysing the correlation matrix and plotted using the first two principal components (PC1 and PC2) on X-axis and Y-axis. All the statistical analyses and graph preparation were done in OriginPro 2018 (OriginLab Corp., Northampton, USA).

Results and discussion

Fungal diversity and SSBF capable yeasts in emao

Among the three dilutions (10⁻³, 10⁻⁵, and 10⁻⁷) inoculated in triplicates to the SDA plates, the dilution 10⁻⁵ showed distinct and well-differentiated fungal colonies with an average of 30.23 × 10⁵ CFU/g. A total of 50 colonies were selected randomly for subsequent sub-culture and pure culture. Based on the colony characters (Supplementary data S3 and S4), ten representative isolates were considered for SSBF assay and molecular identification. The species identification was confirmed if the query sequence showed ≥ 98% homology to the reference sequence(s) in NCBI BLAST homology search. The fungal species so identified in emao were Hyphopichia burtonii (Hbur-FI38, Hbur-FI44, Hbur-FI47 & Hbur-FI68), Saccharomyces cerevisiae (Scer-FI51), Wickerhamomyces anomalus (Wano-FI52), Candida carpophila (Ccar-FI53), Mucor circinelloides (Mcir-FI60), and Saccharomycopsis malanga (Smal-FI77 and Smal-FI84). The relative abundance of the fungal species in descending order was S. malanga (34%) > H. burtonii (28%) > M. circinelloides (16%) > W. anomalus (12%) > S.cerevisiae (6%) > C. carpophila (4%) (Fig. 1). The amylolytic starters used in Asia are generally integrations of different groups of moulds, yeasts, and bacteria [35]. The fungal species we recorded in this study reveals similarity of emao to other Asian rice beer starters reported earlier [36–38]. The starter cultures used in Asian traditional rice beer fermentation are almost similar to each other except the herbs that vary from locality to locality or community to community.

Fig. 1 — Relative abundance of fungal species recorded in *emao*. A total of fifty random colonies were picked from mixed culture plates inoculated with *emao* sample at 10^–5 dilution

In the present study, five of ten fungal representative isolates were found SSBF positive. Therefore, only those SSBF-positive isolates were considered for beer fermentation from rice substrates. In rice beer fermentation, four SSBF-positive isolates viz. Hbur-FI38, Hbur-FI44, Ccar-FI53, and Smal-FI77 produced beer containing 7–10% ethanol, whereas the isolate Smal-FI84 produced beer containing negligible amount (< 1.5%) of ethanol (Tables 1 and 2). The isolates Hbur-FI47 and Hbur-FI68 were found SSBF negative since they failed to saccharify and ferment the rice substrate in the experiment (Table 1). Such strain level differences of Pichia burtonii (synonym: H. burtonii) with ethanol-producing [37, 39, 40] and non-ethanol-producing [41] characters were reported earlier too. The strain Scer-FI51 (S. cerevisiae) grew superficially on the rice substrate but failed to produce ethanol in the present study. Wild S. cerevisiae strains cannot convert starch into sugars due to the lack of amylolytic activity [42]. The ethanol production by S. cerevisiae strains usually eventuates after the amylolysis and liquefaction of the rice substrate accomplished by the glucoamylase and α-amylase enzyme-producing groups in a mixed-culture fermentation platform [43]. Since no saccharifying agent was introduced together with the S. cerevisiae strain in the present study, the limitations of fermentable sugar concentrations might have arrested the Crabtree effect necessary for ethanol formation in this case. Two more SSBF-negative fungal species, W. anomalus and M. circinelloides, recorded in emao corroborate to the earlier reports of these fungi from other Asian traditional amylolytic starters [37, 44, 45]. W. anomalus (Syn. Pichia anomala) is reported as ubiquitous yeast in winemaking and frequently been isolated from grapes and wines. It can grow in high ethanol content (up to 12.5% v/v) and low pH (up to 2.5 or lower) and recently been reported to have some significant roles such as flavour enhancement, reduction of wine spoilage, and haze prevention in wine fermentations [46] and also show lignocellulosic activity [47, 48]. M. circinelloides found in traditional starters is also a saccharifying mould that can degrade cellulose and starch substrates [49, 50] and enhance antioxidant activity in fermented rice [51].

Table 1.

Simultaneous saccharification and beer fermentation (SSBF) assay with the representative fungal strains isolated from a traditional beer starter (Emao) in rice substrate

Strain ID	Species name	NCBI Seq. ID	SSBF assay
Hbur-FI38	Hyphopichia burtonii	MK673884	+ ve
Hbur-FI44	H. burtonii	MK673885	+ ve
Hbur-FI47	H. burtonii	MK673886	− ve
Scer-FI51	Saccharomyces cerevisiae	MK673887	− ve
Wano-FI52	Wickerhamomyces anomalus	MK673888	− ve
Ccar-FI53	Candida carpophila	MK673889	+ ve
Mcir-FI60	Mucor circinelloides	MK673890	− ve
Hbur-FI68	H. burtonii	MK674262	− ve
Smal-FI77	Saccharomycopsis malanga	MK673891	+ ve
Smal-FI84	S. malanga	MK673892	+ ve

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Table 2.

Biochemical and sensory profiles of the rice beers produced using the non-Saccharomyces yeast strains and Emao. SADPPH DPPH radical scavenging activity; AAE acetic acid equivalent, AWS American wine society

Biochemical profile								Sensory profile
Beer sample	Ethanol (% v/v)	Energy (kcal/100 mL)	Ascorbate (mg/100 mL)	Phenol (mg/L)	SADPPH (%)	Reducing power (A₇₀₀₎	Antioxidant activity (g AAE/L)	Appearance (Max = 3) (a)	Aroma (Max = 6) (b)	Taste &texture (Max = 6) (c)	Aftertaste (Max = 3) (d)	Overall impression (Max = 2) (e)	Total sensory score (Max = 12) (f = a + b + c + d + e)	Remarks according to AWS
Emao	17.71 ± 0.94	123.98 ± 0.55	17.87 ± 1.51	405 ± 3	27.94 ± 1.38	0.181 ± 0.01	0.19 ± 0.01	1.8 ± 0.63	4 ± 0.63	3.8 ± 0.4	1.8 ± 0.8	1	12.4 ± 2.46	Good
Hbur-FI38	10.49 ± 0.66	68.17 ± 0.89	20.91 ± 1.1	380 ± 2	37.24 ± 1.12	0.181 ± 0.01	0.15 ± 0.01	2.4 ± 0.8	4.6 ± 0.49	3.6 ± 0.49	2 ± 0.8	2 ± 0.45	13.8 ± 0.3.03	Good
Hbur-FI44	10.23 ± 0.82	67.58 ± 0.46	36.06 ± 1.86	500 ± 3	35.67 ± 0.86	0.346 ± 0.03	0.24 ± 0.01	1.8 ± 0.75	4.6 ± 0.55	3.4 ± 0.8	1.8 ± 0.45	1.2 ± 0.45	12.8 ± 3	Good
Ccar-FI53	9.10 ± 0.78	59.80 ± 0.65	19.39 ± 1.3	230 ± 3	31.8 ± 0.92	0.14 ± 0.04	0.13 ± 0.02	2	3.5 ± 0.55	3 ± 0.8	1	1	10.5 ± 1.35	Commercially acceptable
Smal-FI77	7.08 ± 0.36	54.63 ± 0.36	16.36 ± 1.15	370 ± 1	28.08 ± 1.1	0.154 ± 0.03	0.09 ± 0.02	2.2 ± 0.75	3.6 ± 0.55	2.8 ± 0.45	1.2 ± 0.45	0.80 ± 0.45	10.6 ± 2.65	Commercially acceptable
Smal-FI84	1.14 ± 0.89	102.15 ± 0.74	17.87 ± 1.26	510 ± 2	51.43 ± 0.88	0.166 ± 0.03	0.16 ± 0.01	1	1.2 ± 0.45	2.2 ± 0.45	1.2 ± 0.63	0.8 ± 0.45	6.2 ± 2.08	Deficient

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Physicochemical changes during different stages of beer fermentation

Beer quality is the result of intricate and dynamic interactions in metabolic pathways, both during the brewing process and in the packaged beer [52]. All the metabolic pathways and enzymatic reactions depend on the types of microorganisms involved in a fermentation, which may differ in their metabolic activity in a prevailing condition, thus producing different end products. Some physicochemical parameters have a direct or indirect correlation with the beer quality and thus act as quality indicators, while some of them also reveal the nutritional and nutraceutical potential of the product. The rice beer produced by five SSBF capable yeast strains (Hbur-FI38, Hbur-FI44, Ccar-FI53, Smal-FI77, and Smal-FI84) and the traditional starter (emao) were harvested after 14, 21, and 28 days of fermentation. After 7 days of fermentation, the volume of beer produced in each fermentation bed was insufficient for biochemical analyses of five different parameters (ethanol, TC, TRS, FAN, and pH); therefore, the samples were collected and analysed from 14 days and onwards until 28 days of fermentation.

pH

All the rice beer samples were found acidic, all being within a pH range of 3.03 ± 0.01 to 4.62 ± 0.02 throughout the fermentation period (Fig. 2(d)). A gradual increase in pH with longer fermentation period was observed in all the samples except Smal-FI84. The pH affects beer stability during aging and also colour, freshness, taste, and flavour of the beer [53]. Moreover, low pH (< 4) inactivates some pathogenic coliforms like E. coli and Salmonella Typhimurium [54], ensuring enhanced shelf-life and safety to the products. A similar pH range and gradual increase in pH with longer storage time as observed in this study were also reported by Deka et al. [55] in jou, which might be a feature of ethnic rice beer of the Boro community.

Ethanol

A gradual increase in ethanol concentrations with longer fermentation periods was observed in all the fermentation jars set for in the present study (Fig. 2(a)). The ethanol concentration of the rice beer produced with the traditional starter (emao) was the highest throughout the fermentation period attaining a maximum of 17.7% (v/v) ethanol after 28 days of fermentation. Other traditional Asian rice beer also contain such high ethanol concentrations, viz. Japanese sake with 20% (v/v) ethanol [56], Philippine tapuy with 16–18% (v/v) ethanol [57], and Chinese rice wine even more than 20% (v/v) ethanol [58]. In contrast, the ethanol concentrations of the beer produced with the individual isolates were lower and varied considerably from strain to strain in the present study. The strains, Hbur-FI38 and Hbur-FI44, produced beer containing almost the same concentration of ethanol (~ 10% v/v) which was the highest among the strains followed by Ccar-FI53 (9% v/v ethanol) and Smal-FI77 (7% v/v ethanol) after 28 days of fermentation (Table 2). The ethanol content in beer produced with Smal-FI84 was the lowest and negligible (1% v/v ethanol) though there was a prominent saccharification observed until 28 days of fermentation. It is a consensus that non-Saccharomyces yeasts cannot tolerate ethanol concentrations above 5–7% (v/v) [59, 60], but the present data contradict to that as because four of five strains (Hbur-FI38, Hbur-FI44, Ccar-FI53, and Smal-FI77) produced beer containing above 7% ethanol.

Water is not added in jou fermentation using emao; therefore, it is initially a solid-state fermentation. However, as the amylolysis and liquefaction of rice substrate increase, it slowly becomes semi-solid to semiliquor state fermentation (SSSLF) in the later stages of jou fermentation which is similar to the Chinese rice wine fermentation reported earlier [58]. In Asian traditional rice wine fermentations, generally three major groups of microorganisms viz. saccharifying, ethanol-producing and lactic acid–producing bacteria, are reported earlier. Ethanol concentration increases gradually and the ethanol intolerant microorganisms are eliminated in the later stages of fermentations. Besides, the accumulation of certain secondary inhibitory metabolites like acids, carbonyl compounds, and killer toxins also results in growth inhibition and cell death to several microorganisms including the non-Saccharomyces yeasts in the later stages of fermentation. However, if there is a formation of lower concentrations of inhibitory compounds during co-growing of Saccharomyces and non-Saccharomyces in a mixed fermentation, the co-existence and viability of non-Saccharomyces yeasts increase even at high ethanol content [61].

High ethanol-producing yeast strains possess immense potentiality that can be employed in the growing biofuel industry. However, high concentrations of ethanol in alcoholic beverages often pose several issues like social and health concerns, road safety, and increased tax burdens, [62, 63]. Contrary to adverse health issues related to higher alcohol consumption, chronic low-dose alcohol consumption is cardioprotective [64, 65] and helpful in patients with acute ischemic stroke [66]. We observed much lower ethanol content (ranging from 1–10% v/v) in beers produced by the non-Saccharomyces yeasts in comparison to the beer produced by the traditional starter emao where ethanol content was 17.7% (v/v) after 28 days of fermentation period (Table 2). Mangang et al. [67] observed an alcohol content of 5.71% v/v in a rice beer produced by a mixed culture of P. pentosaceus, A. oryzae, and P. anomala after 9 days of fermentation, that is comparable to the ethanol concentrations of the beer produced by Hbur-FI38 (6.91 ± 0.54% v/v) and Hbur-FI44 (6.56 ± 0.66% v/v) after 14 days of fermentation in the present study (Fig. 2).

Total carbohydrate and total reducing sugars

The rice beer produced by Smal-FI84 showed the highest TC and TRS concentrations among all the beer samples throughout the fermentation period (Fig. 2(b, c)), whereas the lowest TC concentration was observed in the beer produced by Ccar-FI53 (10.06 ± 0.6 g/L). A progressive decrease in the TC concentrations was observed during the fermentation of all the beer samples. During the rice beer fermentation by traditional starters which follows solid fermentation to SSSLF, a continuous flow of fermentable reducing sugars to the liquid state is ensured by the amylolytic microbial groups by breaking the complex carbohydrates packed in the solid-state (rice substrate) into liquefied simple carbohydrates, behaving like a fed-batch system. TRS content reflects the degree of saccharification in rice beer fermentations [51]. However, reducing sugars are also utilised during active fermentation lowering the residual reducing sugar concentrations. Some yeasts including Saccharomyces spp. produce ethanol only in anaerobic condition due to Pasteur effect [68], whereas there are several non-Saccharomayces yeasts [69, 70] and filamentous fungi [71, 72] which can produce ethanol in the presence of oxygen when sugar is present in excess (Crabtree effect). In the present study, a positive correlation between TC and TRS (r = 0.99, p > 0.05) was observed, while there was a negative correlation of ethanol to TC and TRS (Supplementary data S5). In Crabtree-positive yeasts, glucose is completely depleted to form ethanol which is later on utilised as a carbon source [69].

The percent change of ethanol, FAN, TC and TRS in beer samples after 14, 21 and 28 days of fermentation period is shown in Fig. 3. In case of emao where several microbes act together, the TC content (41.87 ± 0.32 g/L) of the beer was observed comparatively high despite of high ethanol content in it after 28 days of fermentation (Fig. 4(b)). Inactivation of the amylolytic microbes or enzymes by increasing ethanol concentration could be a reason for the accumulation of residual complex carbohydrates in the beer produced by emao, because there was a low % change of TC (%ΔTC = − 1.71%) in rice beer collected after 21–28 days of fermentation with emao (Fig. 3). The beer produced using the strains Hbur-FI38, Hbur-FI44, and Hbur-FI53 showed lower %ΔTC during the fermentation period from the 21st to the 28th day than the period from the 14th to the 21st day, exhibiting a decline in amylolytic activity. In the case of Smal-FI84, both %ΔTC and %ΔTRS were low throughout the fermentation period (Fig. 3) revealing a slow utilisation of carbohydrates and a low amylolytic activity by the strain.

Fig. 4 — Physicochemical and nutritional attributes of the 28 days old beers produced with the non-*Saccharomyces* yeast strains and *Emao*. a Ethanol, total protein (TP), total reducing sugar (TRS), and colour. b Total carbohydrate (TC), energy value (EV), and free amino nitrogen (FAN). c Total soluble solids (TSS), free amino acids (FAA), pH and titratable acidity (TA). Results are in the average of triplicates. Error bars represent standard deviation (s.d.). Error bars are not visible when s.d. values are too small

Free amino nitrogen (FAN)

FAN comprises amino acids, small peptides, and ammonium ions that serve as the source of nitrogen for yeast metabolism [73, 74]. In conventional beer production, during malting, grain germination activates proteolytic enzymes, which eventually release the required soluble nitrogen pool to initiate amylolysis [75]. On the other hand, without the involvement of malting, rice beer fermentation is dependent on the proteolytic microbes for the assimilable nitrogen. In the present study, the range of FAN concentrations recorded in the beer samples was from 81.86 ± 0.26 mg/L (Hbur-FI38) to 213.23 mg/L (Ccar-FI53) after 14 days of rice fermentation, from 118.06 ± 0.09 mg/L (Hbur-FI38) to 224.11 ± 0.36 mg/L (Ccar-FI53) after 21 days of rice fermentation, and from 128.71 ± 0.36 mg/L (Smal-FI84) to 231.34 ± 0.28 mg/L (Ccar-FI53) after 28 days of rice fermentation (Fig. 2(e)). The beer samples produced using the strain Ccar-FI53 showed consistently higher FAN concentrations, which signified its high extracellular protease activity. The optimal FAN value considered for beer fermentation is ~ 130 mg/L [76]. FAN concentration in the case of the beer produced by Hbur-FI38 (81.86 ± 0.26 mg/L) and Smal-FI77 (82.73 ± 0.12 mg/L) after 14 days of rice fermentation was far below the minimum FAN concentration (100 mg/L) requirement for normal growth of S. cerevisiae [77], although it increased up to the optimal level in the later stages of the fermentation. Low FAN contents are often associated with incomplete and sluggish fermentation and even restriction of yeast cell growth. Such strains with low FAN requirement might help in fermentation of substrates with lower FAN yield such as black-rice wort, sorghum, and honey [78, 79]. As FAA is a constituent of FAN, a positive correlation (r = 0.77, p > 0.05) was observed between them (Supplementary data S5). Moreover, FAN positively correlated with the ethanol content (r = 0.71, p > 0.05) and also showed a significant positive correlation with TP (r = 0.81, p < 0.05). The correlation between FAN and protein can be made from the perspective of the effect of FAN on yeast cell growth, which in turn affects the ethanol fermentation positively. We observed a periodical increase in FAN concentrations in the rice beer samples produced by the yeast strains and the emao, except for both the strains of S. malanga (Smal-FI77 and Smal-FI84) where there was a rapid decrease in FAN concentrations at the later stages of the fermentation (Fig. 2(e)).

Analyses of nutritional and functional properties of the beer samples

Total protein, free amino acids, and total soluble solids

The protein content in beer originates from the grain used as substrate and the associated microbes [80]. As discussed earlier, the effect of microbial biomass on TP is evident from the significantly positive correlation between ethanol concentration and TP. The highest TP value (18.9 ± 0.45 g/L) was recorded in emao, whereas the values ranged between 11.1 ± 0.56 g/L (Smal-FI84) and 14.7 ± 0.36 g/L (Ccar-FI53) among the yeast strains (Fig. 4(b)) in the present study. Ghosh et al. [53] also observed a similar TP value in four traditional rice beers. The protein content of rice (5–8%) [81] is lower than malted barley (10–12%) [82], whereas the TP content in rice beers was found higher than the beer made from malted barley which ranged from 2.04 ± 0.04 to 5.41 ± 0.12 g/L in 15 Indian beers [23]. Utilisation of rice variety with high water-soluble proteins, low protein fraction in the starch-protein matrix [81], and the accelerated mobilisation of nutrients permitted by the SSSLF process also might have a role in high TP content in the final beer product made from rice substrates.

The FAAs recorded in beer samples produced by Hbur-FI44, Ccar-FI53, and Smal-FI77 were higher (> 4 g/L) than that of the beer produced by emao (3.67 ± 0.65 g/L) (Fig. 4(c)). However, in the case of Hbur-FI38 and Smal-FI84, FAA was relatively lower (< 2 g/L) than the others. Yeast’s physiological state exerts an impact on the fermentation performance, and the wort’s amino acid content plays a central role in regulating the metabolism during fermentation [52]. However, very high amino acid content in the final beer product is undesirable as it promotes haze formation [83].

The TSS determines the texture, density, and viscosity of a beer. Among the beer samples produced by the non-Saccharomyces yeasts and emao, TSS recorded in Smal-FI84 (16.2 ± 0.1°Brix) was the highest followed by emao (10 ± 0.2°Brix) (Fig. 4(c)). TSS primarily indicates the amount of fermentable and non-fermentable sugar concentrations and generally declines with the assimilation of fermentable sugars in the medium [84]. TSS showed a significant positive correlation with TC (r = 0.99, p < 0.05) and TRS (r = 0.96, p < 0.05).

Energy value

One important contribution of lower carbohydrate and ethanol concentrations in beer is the reduced EV, which recently has gained much interest due to growing consciousness towards the adverse effects of high caloric diets. Maximum EV was observed in the beer produced by emao (123.98 ± 0.42 kcal/100 mL) (Fig. 4(a)) in the present study, which is slightly lower to the EV of sake (134 kcal per 100 g) (https://www.caloriescalc.com/alcoholic-beverage-rice-sake/). Ethanol carries higher EV than carbohydrates [85]. Therefore, higher the ethanol content, the higher the EV in alcoholic beverages. We observed lower EV in the beer produced by the non-Saccharomyces strains due to lower ethanol content than in the beer produced by emao (Table 2). The percentage reduction in EV of beer achieved by the strains Hbur-FI38, Hbur-FI44, Ccar-FI53, and Smal-FI77 was 45.02%, 45.5%, 51.77%, and 55.94%, respectively, in comparison to emao.

Titratable acidity

In the present study, TA ranged from 0.56 ± 0.03% TAE (in Hbur-FI44) to 1.44 ± 0.1% TAE (in Smal-FI84) in the beer samples. All the yeast strains, except the strains of S. malanga (Smal-FI77 and Smal-FI84), yielded lower TA than the traditional emao (Fig. 4(c)). Lactic acid bacteria (LAB) are often encountered in traditional rice beer starters [86], which usually contribute to the acidity and mild sour taste in rice beer. A significant negative correlation was observed between TA and overall impression (r = − 0.88, p < 0.05), and the rice beer with lower TA also scored better overall impression values in our study (Table 2, Supplementary data S5). However, no significant correlation was observed between the pH and TA (r = − 0.08, p < 0.05) and it corroborates the earlier report of Li et al. [87]. According to the Australian Wine Research Institute, the pH is not correlated with the concentration of acids present, but is influenced by their ability to dissociate. (https://www.awri.com.au/industry_support/winemaking_resources/frequently_asked_questions/acidity_and_ph/#:~:text=be). Nonetheless, organic acids like lactic acid enhance product shelf-life stability and inhibit contamination [88, 89].