Paneer is a soft variant of cheese obtained by heat and acid coagulant of dairy milk and has a spongy texture, mildly acidic flavor, and milky white color. Paneer is widely consumed in South Asian countries such as Afghanistan, Bangladesh, India, and Pakistan. It is usually served for breakfast or used in culinary dishes [1
]. Paneer contains low amounts of lactose (approximately 2%), and has a high percentage of fat (25%), ash (1.8%), and protein (20%) content, especially when it is prepared from buffalo milk [2
]. Paneer has a high moisture content (52%), providing optimal conditions for bacterial growth, leading to a shorter shelf-life [3
]. The shelf-life of paneer is only one day at room temperature and 7 days in refrigerator temperature [4
]. Many attempts have been made to enhance the shelf life of paneer, such as using different packaging material [4
], use of different spices [5
], addition of plant essential oil [3
], and applying hurdle technology [4
], etc. Production and development of different types of paneer such as soy paneer, paneer from peanut milk, filled paneer, vegetable-impregnated paneer, and ultrafiltration have also been previously attempted [6
In paneer processing, several acids are used for coagulation, such as citric acid, tartaric acid, lactic acid (LA), malic acid, and acetic acid, with heat to break down the casein structure as curd form which is further collected with a muslin cloth and served as fresh paneer product [10
]. Conventionally, paneer is produced using sour fruit juice, such as lemon amla, as a coagulant [10
]. The amount and type of acid plays an important role in paneer quality [8
], and using alternative coagulants such as fermented whey and buttermilk may be effective in producing paneer with a better texture and improved health benefits [11
In this study, we produced paneer from dairy milk mixed with fermented brown rice milk (micro-milled rice slurry) as a coagulant to increase the nutritional value of paneer, and also to produce an acceptable product to utilize rice milk. Studies have shown that brown rice is more nutritious as it contains additional nutrients lacking in white rice and is low in calories compared to white rice [12
] and therefore we micro wet milled (MWM) brown rice to produce rice milk. This system produces rice milk from raw brown rice without prior polishing or cooking. Advantages in this system are that it produces less heat during operation, preserving heat-sensitive nutrients. The MWM system can mill rice into particle sizes less than 2 µm, which can then be used as a rice milk beverage [14
]. The MWM rice milk was subject to LA fermentation to produce a novel coagulant for paneer processing. LA fermentation of rice milk can improve the nutritional value by synthesizing the mineral, vitamins, and amino acid [15
]. In addition, it is expected to improve the paneer texture and reduce the amount of lactose as the demand for lactose-free food is growing globally, due to its health implication for lactose-intolerant people [16
Conventionally, the fermentation of starchy material such as rice is done after a long process of gelatinization, liquefaction and enzymatic saccharification to reducing sugars. Contrarily, saccharification of starch and fermentation can be done simultaneously to avoid energy and time consumption. Studies have shown that simultaneous saccharification and fermentation (SSF) is a simple method for fermentation of starchy material to LA and ethanol [17
The objectives of this study are to incorporate rice milk in paneer making, and also to analyze the appropriate method of saccharification and fermentation for rice milk fermentation, as no study has mentioned the use of fermented rice milk in paneer processing.
2. Materials and Methods
2.1. Sample Collection
Brown rice (Koshihikari) was collected from Ibaraki, Japan, and used for producing rice milk (rice slurry). Dairy whole milk was procured from Takanashi, Iwate, Japan.
2.2. Production of Rice Milk
Rice milk (MWM rice slurry) was prepared from brown rice. Brown rice was soaked in water at a rice to water ratio of 1:2 and kept at 2 °C for 5 h. After soaking, rice was coarsely ground with a mixer (Hamilton Beach-number, Japan) for 3 min and then milled with MWM to produce rice milk according to the method described by Koyama and Kitamura [14
] with slight modifications. The MWM process flow diagram is shown in Figure 1
. The rice coarse slurry mixture was kept in a rotary feeding machine and fed to the milling stone at a rate of 18 g min−1
. Concurrently, the tubing pump fed water at a rate of 40 mL min−1
to the stone mill. The rotational speed of the milling stone was 50 rpm. The stone mill has grooves, an exterior surface area of 207 cm2
, and a radius of 12 cm. The larger contact surface of the mill produces smaller particle sizes and the optimum milling conditions were considered to obtain the smallest particle size. Rice milk was collected simultaneously in the collection tank as the course rice slurry was milled.
2.3. Preparation of Fermented Rice Milk
The raw rice milk was first gelatinized and liquified by 0.1% alpha-amylase enzyme at 90 °C for 30 min. After the liquification process, four different conditions were applied for saccharification and fermentation. The first samples were prepared with simultaneous saccharification and fermentation (SSF) processes, where 0.1% glucoamylase (Glucozyme) and commercial lactic acid bacteria (LAB) starter (0.0016 g) fermenting culture containing Lactobacillus planetarium spp. (Bactoferm® Vegestart 60, CHR HANSEN Biosystems, Horsholm, Denmark) was added to 200 g of rice milk, and saccharification and fermentation were done simultaneously. The second set of samples, saccharification then fermentation (SF), were prepared with the conventional approach, where the rice milk was saccharified prior to fermentation. For SF, rice milk was saccharified with 0.1% glucoamylase and kept at 55 °C for 12 h before fermentation. The third type of samples, without saccharification (WS), were prepared from gelatinized and liquefied rice milk and were fermented directly without saccharification. Fourthly, samples were prepared by adding 0.5% of CaCO3 to SSF for evaluating the effect of pH on rice milk fermentation (CSSF). The same fermentation conditions were used for all samples. Fermentation was carried out in 500 mL Erlenmeyer flasks in a shaker incubator at a speed of 100 rpm for 72 h at 37 °C in triplicates. Samples were collected at 12 h, 24 h, 48 h, and 72 h, and their properties were analyzed.
2.4. Determination of Physical Properties of Rice Milk
The particle size of the rice milk was determined by a laser diffraction particle analyzer (SALD-2200, Shimadzu, Japan) in wet measurement mode. Data were expressed as average particle size (D50). The viscosity of the rice milk was measured using a Brookfield-type viscometer (DV-E, Brookfield Engineering) at 25 °C and at a speed of 12 rpm. Glucose amount was determined by a C2 test kit (Wako Pure Chemical Industries, Osaka, Japan).
2.5. Determination of Total Reducing Sugar, pH, and LA
Total reducing sugar was analyzed with a total reducing sugar kit (Megazyme, Wicklow, Ireland). Samples were diluted with a sample to distilled water ratio of 1:10 ratio and quantified at the absorbance of 340 mm with a spectrophotometer (JASCO V630 Japan). The pH of the samples was determined with a HORIBA scientific pH meter. LA content was measured by titration with 0.05 molL−1
NaOH, according to the method described by Maslanka et al. [18
2.6. Microbial Analysis
Total LAB count and the total microbial count were measured for fermented rice milk and paneer samples using Petrifilm plates (3 M, St. Paul, MN, USA), and plated for aerobic counts using Petrifilm™ Aerobic. Samples were diluted up to 1010
with 0.9% saline solution, then 1 mL of selected dilutions were plated for aerobic counts using Petrifilm™ Aerobic Count plates and 3 M Petri films LAB. The plates were incubated at 37 °C for 48 h then counted for total LAB and total microbial count. The results are presented as log colony-forming unit (CFU) mL−1
of the sample, as instructed by the manufacturer [19
2.7. Development of Paneer
Paneer was prepared with the method stated by Kumar et al. [1
], with slight modifications. Dairy milk was heated to 82 °C for 5 min then cooled to 70 °C. Fermented rice milk was heated to 70 °C and added at the percentage of 10%, 20%, and 30% to dairy milk as a coagulant containing LA to lower the milk pH below the milk protein isoelectric point of 4.6 for successful coagulation [20
]. Milk was continuously stirred until the whey and curd were separated. The coagulated milk was rested for 5 min and then drained using a muslin cloth. The coagulate was then pressed by 4 kg weights for 15 min. Paneer samples were sliced into 20 g cubic shapes and were packaged individually in sterilized zip-lock plastic bags and stored at 4 °C for 12 days. The control samples were prepared with 2% citric acid as a coagulant instead of rice milk.
2.8. Determination of Moisture Content, Total Yield, and Total Acidity of Paneer
The moisture content of the samples was determined by drying method the difference in weights before and after they were oven dried at 105 °C for 24 h, according to official methods [21
].The yield of paneer is the difference of total dairy plus rice milk used and the whey obtained [8
]. Total acidity was measured by titration, where 3 g of sample was diluted in 120 mL distilled water and homogenized with a mixer. Titration was carried out with an automatic potentiometric titrator using 0.05 mol L−1
2.9. Color Measurement of Paneer
The samples from fresh and stored paneer were subjected to color measurements. The picture of the samples were taken with a color scanner (Epson GT-X980) and analyzed using MATLAB software [22
]. The color value was recorded as RGB (red, green, blue) from 0 to 255 values, and was converted to Commission Internationale de l’Elcairage (CIE) color parameter as L*, a*, and b* values using EasyRGB software (IRO group Ltd., Triest, Italy).
2.10. Texture Measurement
The texture of paneer was analyzed with a texture analyzer (EZ-SX with 100 N load cell, Shimadzu, Japan). Compression tests were carried out using a 20 mm cylinder probe with a speed of 1 mm s−1
. Compression results were collected and analyzed using the software (Trapezium 1.4.2) attached to the texture analyzer obtaining the hardness of paneer samples. Hardness is the peak force of the first compression when the probe compressed the sample [23
2.11. Storage Test
A storage test was carried out to determine the physiochemical and biological changes in paneer properties during storage. The paneer samples (0%) rice milk as control with 2% citric acid, 10%, 20%, and 30% v/v rice milk and dairy milk were prepared and stored at 4 °C for 12 days. The samples were evaluated for moisture, microbial, color, and texture properties on days 1, 3, 6, 9, and 12 of storage.
2.12. Sensory Evaluation
Sensory evaluation was determined using a nine-point hedonic scale [24
]. Paneer samples were freshly prepared and stored at 4 °C until the test. The test was done on the same day when paneer samples were prepared. The samples temperature was equilibrated with room temperature before the test. The organoleptic properties were evaluated by 20 semi-trained panelists for appearance, taste, texture, smell, and overall acceptability.
2.13. Statistical Analysis
The experiment was replicated three times, and the data was collected and analyzed with statistical software JMP 2.0.1 (SAS Institute Japan Ltd., Tokyo, Japan). Tukey HSD test α = 0.05 was used to differentiate the differences between analysis means.